Methods and Systems for Processing Polynucleotides

ABSTRACT

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing. In some cases, this disclosure provides methods for the generation of polynucleotide barcode libraries, and for the attachment of such polynucleotides to target polynucleotides.

CROSS-REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/392,557, filed Dec. 28, 2016, which is a continuation ofU.S. patent application Ser. No. 14/104,650, filed Dec. 12, 2013, nowU.S. Pat. No. 9,567,631, which claims priority to U.S. ProvisionalPatent Application No. 61/737,374, filed Dec. 14, 2012, saidapplications are incorporated herein by reference in their entiretiesfor all purposes.

BACKGROUND

Polynucleotide barcodes have utility in numerous applications, includingnext generation sequencing techniques. Such barcodes generally containunique identifier sequences, which can be extremely expensive tomanufacture at sufficient diversity and scale. The cost of synthesizinga single polynucleotide barcode is a function of the cost per baseduring synthesis and the length of the polynucleotide. The cost ofsynthesizing a plurality of barcodes, each with a different sequence, istherefore equivalent to the cost per base, multiplied by the number ofbases per molecule, multiplied by the number of molecules within theplurality of barcodes. Currently, it costs approximately $0.10 per baseto synthesize a DNA sequence. For a barcode library of tens of thousandsto millions of barcodes, this cost is prohibitive. Thus, there is asignificant need for improved methods of generating libraries ofbarcodes.

SUMMARY

This disclosure provides methods, compositions, systems, and kits forthe generation of polynucleotide barcodes and the use of suchpolynucleotide barcodes. Such polynucleotide barcodes may be used forany suitable application.

An aspect of the disclosure provides a library comprising one or morepolynucleotides, each of the polynucleotides comprising a barcodesequence, wherein the polynucleotides are disposed within one or morepartitions, and wherein the library comprises at least about 1,000different barcode sequences.

In some cases, the barcode sequences are at least about 5 nucleotides inlength. Also, the barcode sequences may be random polynucleotidesequences.

Moreover, the partitions may comprise, on average, about 1polynucleotide, about 0.5 polynucleotides, or about 0.1 polynucleotides.The partitions may be droplets, capsules, wells or beads.

Furthermore, the library may comprise at least about 10,000 differentbarcode sequences, at least about 100,000 different barcode sequences,at least about 500,000 different barcode sequences, at least about1,000,000 different barcode sequences, at least about 2,500,00 differentbarcode sequences, at least about 5,000,000 different barcode sequences,at least about 10,000,000, at least about 25,000,000, at least about50,000,000, or at least about 100,000,000 different barcode sequences.

In some cases, the partitions may comprise multiple copies of the samepolynucleotide.

Additionally, each of the polynucleotides may comprise a sequenceselected from the group consisting of an immobilization sequence, anannealing sequence for a sequencing primer, and a sequence compatiblefor ligation with a target polynucleotide.

In some cases, each of the polynucleotides is a MALBAC primer.

Another aspect of the disclosure provides a method of synthesizing alibrary of polynucleotides comprising barcode sequences, the methodcomprising: a.) synthesizing a plurality of polynucleotides comprisingbarcode sequences; b.) separating the polynucleotides into a pluralityof partitions, thereby generating partitioned polynucleotides; c.)amplifying the partitioned polynucleotides, thereby generating amplifiedpolynucleotides; and d.) isolating partitions comprising amplifiedpolynucleotides. In some cases, the synthesizing comprises including amixture of adenine, thymine, guanine, and cytosine in a couplingreaction.

Moreover, the separating may comprise performing a limiting dilution,thereby generating diluted polynucleotides. In some cases, theseparating further comprises partitioning said diluted polynucleotides.

Additionally, the amplifying may be performed by a method selected fromthe group consisting of polymerase chain reaction, asymmetric polymerasechain reaction, emulsion PCR (ePCR), ePCR including the use of a bead,ePCR including the use of a hydrogel, multiple annealing andlooping-based amplification cycles (MALBAC), single primer isothermalamplification, and combinations thereof. In some cases, the amplifyingis performed using an RNA primer and may include exposing the amplifiedpolynucleotides to an RNAase H.

In some cases, each of said polynucleotides comprising barcode sequencesis a MALBAC primer.

In some cases, the isolating may be performed by flow-assisted sorting.

Also, a hairpin structure may be formed from a polynucleotide selectedfrom the group consisting of the polynucleotides comprising barcodesequences and the amplified polynucleotides. In some cases, a method mayfurther comprise cutting the hairpin structure within an unannealedregion.

Moreover, a polynucleotide selected from the group consisting of saidpolynucleotides comprising barcode sequences, said partitionedpolynucleotides, and said amplified polynucleotides may be attached to abead.

The method may further comprise annealing the amplified polynucleotideswith a partially complementary sequence. The partially complementarysequence may comprise a barcode sequence.

The method may further comprise attaching at least one of the amplifiedpolynucleotides to a target sequence. The target sequence may befragmented. In some cases, the target sequence is fragmented by a methodselected from the group consisting of mechanical shear and treatmentwith an enzyme. The mechanical shear may be induced by ultrasound. Insome cases, the enzyme is selected from the group consisting of arestriction enzyme, a fragmentase, and a transposase. Additionally, theattaching may be performed by a method selected from the groupconsisting of ligation and amplification.

In some cases, the amplification is a MALBAC amplification performedwith MALBAC primers, thereby generating a MALBAC amplification product.In some cases, the MALBAC primers comprise the amplifiedpolynucleotides. In some cases, the MALBAC primers comprisepolynucleotides that are not said amplified polynucleotides. In suchcases, the method may further comprise attaching the MALBACamplification product to the amplified polynucleotide.

Additionally, each of the partitions may comprise, on average, about 1polynucleotide comprising a barcode sequence, 0.5 polynucleotidescomprising barcode sequences, or 0.1 polynucleotides comprising barcodesequences. Moreover, the partitions may be selected from the groupconsisting of droplets, capsules, and wells.

In some cases, the library comprises at least about 1,000 differentbarcode sequences, at least about 10,000 different barcode sequences, atleast about 100,000 different barcode sequences, at least about 500,000different barcode sequences, at least about 1,000,000 different barcodesequences, at least about 2,500,00 different barcode sequences, at leastabout 5,000,000 different barcode sequences, at least about 10,000,000,at least about 25,000,000, at least about 50,000,000, or at least about100,000,000 different barcode sequences.

In some cases, the partitions comprise multiple copies of the samepolynucleotide comprising a barcode sequence.

Moreover, the polynucleotides comprising barcode sequences may comprisea sequence selected from the group consisting of an immobilizationsequence, an annealing sequence for a sequencing primer, and a sequencecompatible for ligation with a target polynucleotide.

An additional aspect of the disclosure provides a library comprising atleast about 1,000 beads, wherein each bead of the at least about 1,000beads comprises a different barcode sequence. In some cases, thedifferent barcode sequence can be included in a polynucleotidecomprising an immobilization sequence and/or an annealing sequence for asequencing primer. In some cases, the different barcode sequence can beat least about 5 nucleotides or at least about 10 nucleotides in length.In some cases, the different barcode sequence can be a randompolynucleotide sequence or can be generated combinatorially.

Moreover, each of the 1,000 beads can comprise multiple copies of thedifferent barcode sequence. For example, each of the 1,000 beads maycomprise at least about 100,000, at least about 1,000,000, or at leastabout 10,000,000 copies of the different barcode sequence. In somecases, the library can further comprise two or more beads comprising thesame barcode sequence. In some cases, at least two beads of the 1,000beads can comprise the same barcode sequence. Furthermore, the at leastabout 1,000 beads may comprise at least about 10,000 beads, or at leastabout 100,000 beads.

Also, the library can comprise at least about 1,000, at least about10,000, at least about 100,000, at least about 1,000,000, at least about2,500,000, at least about 5,000,000, at least about 10,000,000, at leastabout 25,000,000, at least about 50,000,000, or at least about100,000,000 different barcode sequences.

In some cases, the at least about 1,000 beads can be distributed acrossa plurality of partitions. In some cases, the partitions can be dropletsof an emulsion. In some cases, each bead of the 1,000 beads can beincluded in a different partition. In some cases, the differentpartition can be a droplet of an emulsion. In some cases, two or morebeads of the 1,000 beads can be included in a different partition. Insome cases, the different partition can be a droplet of an emulsion. Insome cases, the 1,000 beads can be hydrogel beads.

An additional aspect of the disclosure provides for use of a library,composition, method, device, or kit described herein in partitioningspecies, in partitioning oligonucleotides, in stimulus-selective releaseof species from partitions, in performing reactions (e.g., ligation andamplification reactions) in partitions, in performing nucleic acidsynthesis reactions, in barcoding nucleic acid, in preparingpolynucleotides for sequencing, in sequencing polynucleotides, inmutation detection, in neurologic disorder diagnostics, in diabetesdiagnostics, in fetal aneuploidy diagnostics, in cancer mutationdetection and forensics, in disease detection, in medical diagnostics,in low input nucleic acid applications, in circulating tumor cell (CTC)sequencing, in polynucleotide phasing, in sequencing polynucleotidesfrom small numbers of cells, in analyzing gene expression, inpartitioning polynucleotides from cells, or in a combination thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of methods, compositions, systems, and devices ofthis disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of this disclosurewill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of themethods, compositions, systems, and devices of this disclosure areutilized, and the accompanying drawings of which:

FIG. 1 is schematically depicts an example forked adapter.

FIG. 2 schematically depicts example placements of barcode regions.

FIG. 3 depicts example sequences of two forked adapters ligated toopposite ends of a target polynucleotide. Full-length sequence disclosedas SEQ ID NO: 35.

FIG. 4 is a schematic example method used to generate a forked adapterdescribed in Example 1.

FIG. 5 is a schematic example of a capsule within a capsule described inExample 2.

FIG. 6 is a schematic example of capsules within a capsule described inExample 3.

FIG. 7 is a schematic example of a product (or intermediate) that may begenerated according to methods of Example 4.

FIGS. 8a-c depict example sequences described in Example 4.

FIGS. 9a-j depict example sequences described in Example 5.

FIGS. 10a-e depict example sequences described in Example 6.

FIGS. 11a-d schematically depict methods and structures described inExample 7.

FIG. 12 schematically depicts the production capsules via an exampleflow-focusing method.

FIG. 13 schematically depicts the production of capsules within capsulesvia an example flow-focusing method.

FIGS. 14a-e schematically depict methods and structures described inExample 8.

FIGS. 15a-e schematically depict methods and structures described inExample 9.

FIG. 16 schematically depicts methods and structures described inExample 10.

FIG. 17 schematically depicts a capsule within a capsule described inExample 11.

FIG. 18 schematically depicts capsules within a capsule described inExample 12.

FIGS. 19a-e depict example sequences described in Example 13. FIG. 19fdescribes example methods and structures described in Example 13.

FIG. 20 schematically depicts a capsule within a capsule described inExample 14.

FIGS. 21a-c schematically depict methods and structures described inExample 15.

FIG. 22 schematically depicts a capsule within a capsule described inExample 16.

FIG. 23 schematically depicts a capsule within a capsule described inExample 17.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

This disclosure provides methods, compositions, systems, and kits forthe generation of polynucleotide barcodes and the use of suchpolynucleotide barcodes. Such polynucleotide barcodes may be used forany suitable application. In some cases, the polynucleotide barcodesprovided in this disclosure may be used in next generation sequencingreactions. Next generation sequencing reactions include the sequencingof whole genomes, detection of specific sequences such as singlenucleotide polymorphisms (SNPs) and other mutations, detection ofnucleic acid (e.g., deoxyribonucleic acid) insertions, and detection ofnucleic acid deletions.

Utilization of the methods, compositions, systems, and kits describedherein may incorporate, unless otherwise indicated, any conventionaltechniques of organic chemistry, polymer technology, microfluidics,molecular biology, recombinant techniques, cell biology, biochemistry,and immunology. Such conventional techniques include well and microwellconstruction, capsule generation, generation of emulsions, spotting,microfluidic device construction, polymer chemistry, restrictiondigestion, ligation, cloning, polynucleotide sequencing, andpolynucleotide sequence assembly. Specific, non-limiting, illustrationsof suitable techniques are described throughout this disclosure.However, equivalent procedures may also be utilized. Descriptions ofcertain techniques may be found in standard laboratory manuals, such asGenome Analysis: A Laboratory Manual Series (Vols. I-IV), UsingAntibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer:A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (allfrom Cold Spring Harbor Laboratory Press), and “OligonucleotideSynthesis: A Practical Approach” 1984, IRL Press London, all of whichare herein incorporated in their entirety by reference for all purposes.

I. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including,”“includes,” “having,” “has,” “with,” “such as,” or variants thereof, areused in either the specification and/or the claims, such terms are notlimiting and are intended to be inclusive in a manner similar to theterm “comprising”.

The term “about,” as used herein, generally refers to a range that is15% greater than or less than a stated numerical value within thecontext of the particular usage. For example, “about 10” would include arange from 8.5 to 11.5.

The term “barcode,” as used herein, generally refers to a label that maybe attached to an analyte to convey information about the analyte. Forexample, a barcode may be a polynucleotide sequence attached tofragments of a target polynucleotide contained within a particularpartition. This barcode may then be sequenced with the fragments of thetarget polynucleotide. The presence of the same barcode on multiplesequences may provide information about the origin of the sequence. Forexample, a barcode may indicate that the sequence came from a particularpartition and/or a proximal region of a genome. This may be particularlyuseful for sequence assembly when several partitions are pooled beforesequencing.

The term “bp,” as used herein, generally refers to an abbreviation for“base pairs”.

The term “microwell,” as used herein, generally refers to a well with avolume of less than 1 mL. Microwells may be made in various volumes,depending on the application. For example, microwells may be made in asize appropriate to accommodate any of the partition volumes describedherein.

The term “partition,” as used herein, may be a verb or a noun. When usedas a verb (e.g., “to partition,” or “partitioning”), the term generallyrefers to the fractionation (e.g., subdivision) of a species or sample(e.g., a polynucleotide) between vessels that can be used to sequesterone fraction (or subdivision) from another. Such vessels are referred tousing the noun “partition.” Partitioning may be performed, for example,using microfluidics, dilution, dispensing, and the like. A partition maybe, for example, a well, a microwell, a hole, a droplet (e.g., a dropletin an emulsion), a continuous phase of an emulsion, a test tube, a spot,a capsule, a bead, a surface of a bead in dilute solution, or any othersuitable container for sequestering one fraction of a sample fromanother. A partition may also comprise another partition.

The terms “polynucleotide” or “nucleic acid,” as used herein, generallyrefer to molecules comprising a plurality of nucleotides. Exemplarypolynucleotides include deoxyribonucleic acids, ribonucleic acids, andsynthetic analogues thereof, including peptide nucleic acids.

The term “species,” as used herein, generally refers to any substancethat can be used with the methods, compositions, systems, devices, andkits of this disclosure. Examples of species include reagents, analytes,cells, chromosomes, tagging molecules or groups of molecules, barcodes,and any sample comprising any of these species. Any suitable species maybe used, as more fully discussed elsewhere in this disclosure.

II. Polynucleotide Barcoding

Certain applications, for example polynucleotide sequencing, may rely onunique identifiers (“barcodes”) to identify the origin of a sequenceand, for example, to assemble a larger sequence from sequencedfragments. Therefore, it may be desirable to add barcodes topolynucleotide fragments before sequencing. Barcodes may be of a varietyof different formats, including polynucleotide barcodes. Depending uponthe specific application, barcodes may be attached to polynucleotidefragments in a reversible or irreversible manner. Additionally, barcodesmay allow for identification and/or quantification of individualpolynucleotide fragments during sequencing.

Barcodes may be loaded into partitions so that one or more barcodes areintroduced into a particular partition. In some cases, each partitionmay contain a different set of barcodes. This may be accomplished bydirectly dispensing the barcodes into the partitions, or by placing thebarcodes within a partition that is contained within another partition.

The barcodes may be loaded into the partitions at an expected orpredicted ratio of barcodes per species to be barcoded (e.g.,polynucleotide fragment, strand of polynucleotide, cell, etc.). In somecases, the barcodes are loaded into partitions such that about 0.0001,0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000,10000, or 200000 barcodes are loaded per species. In some cases, thebarcodes are loaded into partitions such that more than about 0.0001,0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000,10000, or 200000 barcodes are loaded per species. In some cases, thebarcodes are loaded in the partitions so that less than about 0.0001,0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000,10000, or 200000 barcodes are loaded per species.

When more than one barcode is present per polynucleotide fragment, suchbarcodes may be copies of the same barcode, or may be differentbarcodes. For example, the attachment process may be designed to attachmultiple identical barcodes to a single polynucleotide fragment, ormultiple different barcodes to the polynucleotide fragment.

The methods provided herein may comprise loading a partition with thereagents necessary for the attachment of barcodes to polynucleotidefragments. In the case of ligation reactions, reagents includingrestriction enzymes, ligase enzymes, buffers, adapters, barcodes and thelike may be loaded into a partition. In the case of barcoding byamplification, reagents including primers, DNA polymerases, dNTPs,buffers, barcodes and the like may be loaded into a partition. In thecase of transposon-mediated barcoding (e.g., NEXTERA), reagentsincluding a transposome (i.e., transposase and transposon end complex),buffers, and the like may be loaded into a partition. In the case ofMALBAC-mediated barcoding, reagents including a MALBAC primer, buffers,and the like may be loaded into a partition. As described throughoutthis disclosure, these reagents may be loaded directly into thepartition, or via another partition.

Barcodes may be ligated to a polynucleotide fragment using sticky orblunt ends. Barcoded polynucleotide fragments may also be generated byamplifying a polynucleotide fragment with primers comprising barcodes.In some cases, MALBAC amplification of the polynucleotide fragment maybe used to generate a barcoded polynucleotide fragment. A primer usedfor MALBAC may or may not comprise a barcode. In cases where a MALBACprimer does not comprise a barcode, the barcode may be added to MALBACamplification products by other amplification methods, such as, forexample, PCR. Barcoded polynucleotide fragments may also be generatedusing transposon-mediated methods. As with any other species discussedin this disclosure, these modules may be contained within the same ordifferent partitions, depending on the needs of the assay or process.

In some cases, barcodes may be assembled combinatorially, from smallercomponents designed to assemble in a modular format. For example, threemodules, 1A, 1B, and 1C may be combinatorially assembled to producebarcode 1ABC. Such combinatorial assembly may significantly reduce thecost of synthesizing a plurality of barcodes. For example, acombinatorial system consisting of 3 A modules, 3 B modules, and 3 Cmodules may generate 3*3*3=27 possible barcode sequences from only 9modules.

In some cases, as further described elsewhere in this disclosure,barcodes may be combinatorially assembled by mixing two oligonucleotidesand hybridizing them to produce annealed or partially annealedoligonucleotides (e.g., forked adapters). These barcodes may comprise anoverhang of one or more nucleotides, in order to facilitate ligationwith polynucleotide fragments that are to be barcoded. In some cases,the 5′ end of the antisense strand may be phosphorylated in order toensure double-stranded ligation. Using this approach, different modulesmay be assembled by, for example, mixing oligonucleotides A and B, A andC, A and D, B and C, B, and D, and so on. As described in more detailelsewhere in this disclosure, the annealed oligonucleotides may also besynthesized as a single molecule with a hairpin loop that may be cutafter ligation to the polynucleotide to be barcoded.

As described in more detail elsewhere in this disclosure, attachment ofpolynucleotides to each other may rely on hybridization-compatibleoverhangs. For example, the hybridization between A and T is often usedto ensure ligation compatibility between fragments. In some cases, an Aoverhang may be created by treatment with an enzyme, such as a Taqpolymerase. In some cases, a restriction enzyme may be used to create acleavage product with a single base 3′ overhang which may be, forexample, A or T. Examples of restriction enzymes that leave a singlebase 3′ overhang include MnII, HphI, Hpy188I, HpyAV, HpyCH4III, MboII,BciVI, BmrI, AhdI, and XcmI. In other cases, different overhangs (e.g.,5′ overhangs, overhangs of greater than a single base) may be generatedby restriction enzymes. Additional restriction enzymes that may be usedto generate overhangs include BfuCl, Taq^(α)I, BbVI, Bccl, BceAl, BcoDI,BsmAI, and BsmFI.

III. Generation of Partitioned Barcode Libraries

In some cases, this disclosure provides methods for the generation ofpartitioned barcode libraries and libraries produced according to suchmethods. In some cases, the methods provided herein combine randomsynthesis of DNA sequences, separation into partitions, amplification ofseparated sequences, and isolation of amplified separated sequences toprovide a library of barcodes contained within partitions.

a. Random Synthesis of Polynucleotide Barcodes

In some cases, the methods described herein utilize random methods ofpolynucleotide synthesis, including random methods of DNA synthesis.During random DNA synthesis, any combination of A, C, G, and/or T may beadded to a coupling step so that each type of base in the coupling stepis coupled to a subset of the product. If A, C, G, and T are present atequivalent concentrations, approximately one-quarter of the product willincorporate each base. Successive coupling steps, and the random natureof the coupling reaction, enable the generation of 4^(n) possiblesequences, where n is the number of bases in the polynucleotide. Forexample, a library of random polynucleotides of length 6 could have adiversity of 4⁶=4,096 members, while a library of length 10 would havediversity of 1,048,576 members. Therefore, very large and complexlibraries can be generated. These random sequences may serve asbarcodes.

Any suitable synthetic bases may also be used with the invention. Insome cases, the bases included in each coupling step may be altered inorder to synthesize a preferred product. For example, the number ofbases present in each coupling step may be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more. In some cases, the number of bases present in each couplingstep may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In somecases, the number of bases present in each coupling step may be lessthan 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The concentration of the individual bases may also be altered in orderto synthesize the preferred product. For example, any base may bepresent at a concentration of about 0.1, 0.5, 1, 5, or 10-fold theconcentration of another base. In some cases, any base may be present ata concentration of at least about 0.1, 0.5, 1, 5, or 10-fold theconcentration of another base. In some cases, any base may be present ata concentration of less than about 0.1, 0.5, 1, 5, or 10-fold theconcentration of another base.

The length of the random polynucleotide sequence may be any suitablelength, depending on the application. In some cases, the length of therandom polynucleotide sequence may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some cases, thelength of the random polynucleotide sequence may be at least 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides.In some cases, the length of the random polynucleotide sequence may beless than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides.

In some cases, the library is defined by the number of members. In somecases, a library may comprise about 256, 1024, 4096, 16384, 65536,262144, 1048576, 4194304, 16777216, 67108864, 268435456, 1073741824,4294967296, 17179869184, 68719476736, 2.74878*10¹¹, or 1.09951*10¹²members. In some cases, a library may comprise at least about 256, 1024,4096, 16384, 65536, 262144, 1048576, 4194304, 16777216, 67108864,268435456, 1073741824, 4294967296, 17179869184, 68719476736,2.74878*10¹¹, or 1.09951*10¹² members. In some cases, a library maycomprise less than about 256, 1024, 4096, 16384, 65536, 262144, 1048576,4194304, 16777216, 67108864, 268435456, 1073741824, 4294967296,17179869184, 68719476736, 2.74878*10¹¹, or 1.09951*10¹² members. In somecases, the library is a barcode library. In some cases, a barcodelibrary may comprise at least about 1000, 10000, 100000, 1000000,2500000, 5000000, 10000000, 25000000, 50000000, or 100000000 differentbarcode sequences.

The random barcode libraries may also comprise other polynucleotidesequences. In some cases, these other polynucleotide sequences arenon-random in nature and include, for example, primer binding sites,annealing sites for the generation of forked adapters, immobilizationsequences, and regions that enable annealing with a targetpolynucleotide sequence, and thus barcoding of the polynucleotidesequence.

b. Separation of Polynucleotides into Partitions

After synthesis of polynucleotides comprising random barcode sequences,the polynucleotides are partitioned into separate compartments togenerate a library of partitioned polynucleotides comprising barcodesequences. Any suitable method of separation and any suitable partitionor partitions within partitions may be used.

In some cases, partitioning is performed by diluting the mixture ofpolynucleotides comprising random barcode sequences such that aparticular volume of the dilution contains, on average, less than asingle polynucleotide. The particular volume of the dilution may then betransferred to a partition. In any plurality of partitions, eachpartition is therefore likely to have one or zero polynucleotidemolecules.

In some cases a dilution may be performed such that each partitioncomprises about 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, or more molecules. In some cases a dilution may be performedsuch that each partition comprises at least about 0.001, 0.01, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, or more molecules. In somecases a dilution may be performed such that each partition comprisesless than about 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, or 2 molecules.

In some cases, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe partitions comprise the specified number of molecules. In somecases, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe partitions comprise the specified number of molecules. In somecases, less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe partitions comprise the specified number of molecules.

In some cases, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe partitions comprise one or fewer polynucleotides. In some cases, atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of thepartitions comprise one or fewer polynucleotides. In some cases, lessthan about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of thepartitions comprise one or fewer polynucleotides.

In some cases, a partition is a well, a microwell, a hole, a droplet(e.g., a droplet in an emulsion), a continuous phase of an emulsion, atest tube, a spot, a capsule, a surface of a bead, or any other suitablecontainer for sequestering one fraction of a sample from another. Incases where a partition includes a bead, a primer for amplification maybe attached to the bead. Partitions are described in greater detailelsewhere in this disclosure.

c. Amplification of Partitioned Polynucleotides

The polynucleotides partitioned as described above are then amplified inorder to generate sufficient material for barcoding of a targetpolynucleotide sequence. Any suitable method of amplification may beutilized, including polymerase chain reaction (PCR), ligase chainreaction (LCR), helicase-dependent amplification, linear after theexponential PCR (LATE-PCR), asymmetric amplification, digital PCR,degenerate oligonucleotide primer PCR (DOP-PCR), primer extensionpre-amplification PCR (PEP-PCR), ligation mediated PCR, rolling circleamplification, multiple displacement amplification (MDA), and singleprimer isothermal amplification (SPIA), emulsion PCR (ePCR), ePCRincluding the use of a bead, ePCR including the use of a hydrogel,multiple annealing and looping-based amplification cycles (MALBAC), andcombinations thereof. MALBAC methods are described, for example, in Zonget al., Science, 338(6114), 1622-1626 (2012), which is incorporatedherein by reference, in its entirety.

In some cases, amplification methods that generate single-strandedproduct (e.g., asymmetric amplification, SPIA, and LATE-PCR) may bepreferred, for example. In some cases, amplification methods thatgenerate double-stranded products (e.g., standard PCR) may be preferred.In some cases, an amplification method will exponentially amplify thepartitioned polynucleotide. In some cases, an amplification method willlinearly amplify the partitioned polynucleotide. In some cases, anamplification method will first exponentially and then linearly amplifya polynucleotide. Moreover, a single type of amplification may be usedto amplify polynucleotides or amplification may be completed withsequential steps of different types of amplification. For example, ePCRmay be combined with further rounds of ePCR or may be combined with adifferent type of amplification.

Amplification is performed until a suitable amount of polynucleotidecomprising a barcode is produced. In some cases, amplification may beperformed for 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or morecycles. In some cases, amplification may be performed for at least 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more cycles. In some cases,amplification may be performed for less than 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 cycles.

In some cases, amplification may be performed until a certain amount ofpolynucleotide product is produced in each partition. In some cases,amplification is performed until the amount of polynucleotide product isabout 10,000,000,000; 5,000,000,000; 1,000,000,000; 500,000,000;100,000,000; 50,000,000; 10,000,000; 5,000,000; 1,000,000; 500,000;400,000; 300,000; 200,000; or 100,000 molecules. In some cases,amplification is performed until the amount of polynucleotide product isat least about 100,000; 200,000; 300,000; 400,000; 500,000; 1,000,000;5,000,000; 10,000,000; 50,000,000; 100,000,000; 500,000,000;1,000,000,000; 5,000,000,000; or 10,000,000,000 molecules. In somecases, amplification is performed until the amount of polynucleotideproduct is less than about 10,000,000,000; 5,000,000,000; 1,000,000,000;500,000,000; 100,000,000; 50,000,000; 10,000,000; 5,000,000; 1,000,000;500,000; 400,000; 300,000; 200,000; or 100,000 molecules.

d. Isolation of Partitions Comprising Amplified Sequences

As described above, in some cases polynucleotides comprising barcodesare partitioned such that each partition contains, on average, less thanone polynucleotide sequence. Therefore, in some cases, a fraction of thepartitions will not contain a polynucleotide and therefore cannotcontain an amplified polynucleotide. Thus, it may be desirable toseparate partitions comprising polynucleotides from partitions notcomprising polynucleotides.

In one case, partitions comprising polynucleotides are separated frompartitions not comprising polynucleotides using flow-based sortingmethods capable of identifying partitions comprising polynucleotides. Insome cases an indicator of the presence of a polynucleotide may be usedin order to differentiate partitions comprising polynucleotides fromthose not comprising polynucleotides.

In some cases, a nucleic acid stain may be used to identify partitioncomprising polynucleotides. Exemplary stains include intercalating dyes,minor-groove binders, major groove binders, external binders, andbis-intercalators. Specific examples of such dyes include SYBR green,SYBR blue, DAPI, propidium iodide, SYBR gold, ethidium bromide,acridines, proflavine, acridine orange, acriflavine, fluorcoumanin,ellipticine, daunomycin, chloroquine, distamycin D, chromomycin,homidium, mithramycin, ruthenium polypyridyls, anthramycin,phenanthridines and acridines, ethidium bromide, propidium iodide,hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidiummonoazide, ACMA, indoles, imidazoles (e.g., Hoechst 33258, Hoechst33342, Hoechst 34580 and DAPI), acridine orange (also capable ofintercalating), 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOXBlue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1,TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1,BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1,YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBRGreen II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13,-16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81,-80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, and-63 (red).

In some cases, isolation methods such as magnetic separation orsedimentation of particles may be used. Such methods may include, forexample, a step of attaching a polynucleotide to be amplified, a primercorresponding to said polynucleotide to be amplified, and/or apolynucleotide product of amplification to a bead. In some cases,attachment of a polynucleotide to be amplified, primer corresponding tosaid polynucleotide to be amplified, and/or a polynucleotide product toa bead may be via a photolabile linker, such as, for example, PC AminoC6. In cases where a photolabile linker is used, light may be used torelease a linked polynucleotide from the bead. The bead may be, forexample, a magnetic bead or a latex bead. The bead may then enableseparation by, for example, magnetic sorting or sedimentation.Sedimentation of latex particles may be performed, for example, bycentrifugation in a liquid that is more dense than latex, such asglycerol. In some cases, density gradient centrifugation may be used.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm,500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. A bead may have adiameter of at least about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm,600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. In some cases, a bead may havea diameter of less than about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm,500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. In some cases, a beadmay have a diameter of about 0.001 μm to 1 mm, 0.01 μm to 900 μm, 0.1 μmto 600 μm, 100 μm to 200 μm, 100 μm to 300 μm, 100 μm to 400 μm, 100 μmto 500 μm, 100 μm to 600 μm, 20 μm to 50 μm, 150 μm to 200 μm, 150 μm to300 μm, or 150 μm to 400 μm.

In some cases, a differential charge between the partitions comprisingpolynucleotides and partitions not comprising polynucleotides may beused to isolate partitions comprising polynucleotides, for example byperforming electrophoresis or dielectrophoresis on the partitions.

In some cases, selective swelling or shrinking of partitions, based ondifferences in the osmotic pressures, may be used to identify particlescomprising polynucleotides. In some instances, partitions comprisingpolynucleotides may be isolated by flow fractionation, solventextraction, differential melting (e.g., with nucleic acid probes), orfreezing.

Isolation of partitions comprising polynucleotides provides a library ofpartitioned polynucleotide barcodes with significant diversity whileincurring only a one-time bulk synthesis expense.

IV. Generation of Adapters Comprising Barcodes

The barcodes described in this disclosure can have a variety ofstructures. In some cases, barcodes of this disclosure are a part of anadapter. Generally, an “adapter” is a structure used to enableattachment of a barcode to a target polynucleotide. An adapter maycomprise, for example, a barcode, polynucleotide sequence compatible forligation with a target polynucleotide, and functional sequences such asprimer binding sites and immobilization regions.

In some cases, an adapter is a forked adapter. An example of a forkedadapter is schematically depicted in FIG. 1. With reference to FIG. 1,two copies of a forked adapter structure 106 are depicted on oppositesides of a target polynucleotide 105. Each forked adapter comprises afirst immobilization region 101, a second immobilization region 102, afirst sequencing primer region 103, a second sequencing primer region104 and a pair of partially complementary regions (within 103 and 104)that anneal to each other. Either the sequencing primer regions orimmobilization regions may be used to immobilize the barcodedpolynucleotides, for example, onto the surface of a bead. The sequencingprimer regions may be used, for example, as annealing sites forsequencing primers. In some cases, an overhang may be designed to enablecompatibility with a target sequence. In FIG. 1, the pair of annealedpolynucleotides 103 and 104 have a 3′-T overhang, which is compatiblewith the 3′-A overhang on the target polynucleotide 105. A barcode maybe included in any suitable portion of a forked adapter. Afterattachment of the forked adapter comprising the barcode to the targetsequence 105, the sequencing primer regions 103 and 104 can be used tosequence the target polynucleotide. Another example of a forked adapterstructure includes those used in Illumina™ library preparations andNEBNext® Multiplex Oligos for Illumina available from New EnglandBiolabs™. Examples of non-forked adapters include those disclosed inMerriman et al., Electrophoresis, 33(23) 3397-3417 (2012), which isincorporated herein by reference, in its entirety.

FIG. 2 illustrates three schematic examples of placement of barcoderegions within the forked adapter depicted in FIG. 1. In one example, abarcode 205 (BC1) is placed within the first immobilization region 201or between the first immobilization region 201 and the first sequencingprimer region 203. In another example, a barcode 206 (BC2) is placedwithin or adjacent to the first sequencing primer region 203. In yetanother example, a barcode 207 (BC3) is placed within the secondimmobilization region 202 or between the second immobilization region202 and the second sequencing primer region 204. Although FIG. 2 depictsbarcodes on both ends of the target sequence, this is not necessary, asonly one barcode per target sequence is sufficient for someapplications. However, as described elsewhere in this disclosure, morethan one barcode per target sequence may also be used.

FIG. 3 provides exemplary sequences (SEQ ID NO: 1 and SEQ ID NO: 22) oftwo forked adapters ligated to opposite ends of a target polynucleotide(NNN) and shows barcode regions of each forked adapter at the sequencelevel (bolded, nucleotides 30-37, 71-77, 81-87, and 122-129). In FIG. 3,nucleotides 1-29 represent an immobilization region of the first forkedadapter, nucleotides 38-70 represent a sequencing primer region of thefirst forked adapter, nucleotides 78-80 (NNN) represent a targetpolynucleotide of arbitrary length, nucleotides 88-120 represent asequencing primer region of the second forked adapter, and nucleotides129-153 represent an immobilization region of the second forked adapter.

V. Partitions

a. General Characteristics of Partitions

As described throughout this disclosure, certain methods, compositions,systems, devices, and kits of the disclosure may utilize the subdivision(partitioning) of certain species into separate partitions. A partitionmay be, for example, a well, a microwell, a hole, a droplet (e.g., adroplet in an emulsion), a continuous phase of an emulsion, a test tube,a spot, a capsule, a surface of a bead, or any other suitable containerfor sequestering one fraction of a sample or a species. Partitions maybe used to contain a species for further processing. For example, if aspecies is a polynucleotide analyte, further processing may comprisecutting, ligating, and/or barcoding with species that are reagents. Anynumber of devices, systems or containers may be used to hold, support orcontain partitions. In some cases, a microwell plate may be used tohold, support, or contain partitions. Any suitable microwell plate maybe used, for example microwell plates having 96, 384, or 1536 wells.

Each partition may also contain, or be contained within any othersuitable partition. For example, a well, microwell, hole, a surface of abead, or a tube may comprise a droplet (e.g., a droplet in an emulsion),a continuous phase in an emulsion, a spot, a capsule, or any othersuitable partition. A droplet may comprise a capsule, bead, or anotherdroplet. A capsule may comprise a droplet, bead, or another capsule.These descriptions are merely illustrative, and all suitablecombinations and pluralities are also envisioned. For example, anysuitable partition may comprise a plurality of the same or differentpartitions. In one example, a well or microwell comprises a plurality ofdroplets and a plurality of capsules. In another example, a capsulecomprises a plurality of capsules and a plurality of droplets. Allcombinations of partitions are envisioned. Table 1 shows non-limitingexamples of partitions that may be combined with each other.

TABLE 1 Examples of partitions that may be combined with each other.Well Spot Droplet Capsule Well Well inside Spot inside Droplet Capsulewell well inside well inside well Spot Spot inside Spot inside DropletCapsule well spot inside spot inside spot Droplet Droplet DropletDroplet Droplet inside well inside spot inside droplet inside capsuleCapsule inside droplet Capsule Capsule Capsule Capsule Capsule insidewell inside spot inside droplet inside Spot inside Droplet capsulecapsule inside capsule Surface of a Bead inside Spot on bead Bead insideBead inside Bead well Bead inside droplet capsule spot

Any partition described herein may comprise multiple partitions. Forexample, a partition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10000, or 50000 partitions. A partition may comprise atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000partitions. In some cases, a partition may comprise less than 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000 partitions. In somecases, each partition may comprise 2-50, 2-20, 2-10, or 2-5 partitions.

A partition may comprise any suitable species or mixture of species. Forexample, in some cases a partition may comprise a reagent, an analyte, asample, a cell, and combinations thereof. A partition comprising otherpartitions may comprise certain species in the same partitions andcertain species in different partitions. Species may be distributedbetween any suitable partitions, depending on the needs of theparticular process. For example, any of the partitions in Table 1 maycontain at least one first species and any of the partitions in Table 1may contain at least one second species. In some cases the first speciesmay be a reagent and the second species may be an analyte.

In some cases, a species is a polynucleotide isolated from a cell. Forexample, in some cases polynucleotides (e.g., genomic DNA, RNA, etc.) isisolated from a cell utilizing any suitable method (e.g., a commerciallyavailable kit). The polynucleotide may be quantified. The quantifiedpolynucleotide may then be partitioned into a plurality of partitions asdescribed herein. The partitioning of the polynucleotide may beperformed at a predetermined coverage amount, according to thequantification and the needs of the assay. In some cases, all or most(e.g., at least 50%, 60%, 70%, 80%, 90%, or 95%) of the partitions donot comprise polynucleotides that overlap, such that separate mixturesof non-overlapping fragments are formed across the plurality ofpartitions. The partitioned polynucleotides may then be treatedaccording to any suitable method known in the art or described in thisdisclosure. For example, the partitioned polynucleotides may befragmented, amplified, barcoded, and the like.

Species may be partitioned using a variety of methods. For example,species may be diluted and dispensed across a plurality of partitions. Aterminal dilution of a medium comprising species may be performed suchthat the number of partitions exceeds the number of species. Dilutionmay also be used prior to forming an emulsion or capsules, or prior tospotting a species on a substrate. The ratio of the number of species tothe number of partitions may be about 0.1, 0.5, 1, 2, 4, 8, 10, 20, 50,100, or 1000. The ratio of the number of species to the number ofpartitions may be at least about 0.1, 0.5, 1, 2, 4, 8, 10, 20, 50, 100,or 1000. The ratio of the number of species to the number of partitionsmay be less than about 0.1, 0.5, 1, 2, 4, 8, 10, 20, 50, 100, or 1000.The ratio of the number of species to the number of partitions may rangefrom about 0.1-10, 0.5-10, 1-10, 2-10, 10-100, 100-1000, or more.

Partitioning may also be performed using piezoelectric dropletgeneration (e.g., Bransky et al., Lab on a Chip, 2009, 9, 516-520) orsurface acoustic waves (e.g., Demirci and Montesano, Lab on a Chip,2007, 7, 1139-1145).

The number of partitions employed may vary depending on the application.For example, the number of partitions may be about 5, 10, 50, 100, 250,500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, 20000, 30000,40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000,400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000,3,000,000, 4,000,000, 5,000,000, 10000000, 20000000, or more. The numberof partitions may be at least about 5, 10, 50, 100, 250, 500, 750, 1000,1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000,70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000,700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000,5,000,000, 10000000, 20000000, or more. The number of partitions may beless than about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500,5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000,900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000,20000000. The number of partitions may be about 5-10000000, 5-5000000,5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000, 1,000-5,000,1,000-4,000, 1,000-3,000, or 1,000-2,000.

The number of different barcodes or different sets of barcodes that arepartitioned may vary depending upon, for example, the particularbarcodes to be partitioned and/or the application. Different sets ofbarcodes may be, for example, sets of identical barcodes where theidentical barcodes differ between each set. Or different sets ofbarcodes may be, for example, sets of different barcodes, where each setdiffers in its included barcodes. For example, about 1, 5, 10, 50, 100,1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, ormore different barcodes or different sets of barcodes may bepartitioned. In some examples, at least about 1, 5, 10, 50, 100, 1000,10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000,200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000,1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000,9000000, 10000000, 20000000, 50000000, 100000000, or more differentbarcodes or different sets of barcodes may be partitioned. In someexamples, less than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000,40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000,400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000,3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000,20000000, 50000000, or 100000000 different barcodes or different sets ofbarcodes may be partitioned. In some examples, about 1-5, 5-10, 10-50,50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 barcodes may bepartitioned.

Barcodes may be partitioned at a particular density. For example,barcodes may be partitioned so that each partition contains about 1, 5,10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000, 500,000,600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000,5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,50000000, or 100000000 barcodes per partition. Barcodes may bepartitioned so that each partition contains at least about 1, 5, 10, 50,100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, ormore barcodes per partition. Barcodes may be partitioned so that eachpartition contains less than about 1, 5, 10, 50, 100, 1000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,10000000, 20000000, 50000000, or 100000000 barcodes per partition.Barcodes may be partitioned such that each partition contains about 1-5,5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 barcodes perpartition.

Barcodes may be partitioned such that identical barcodes are partitionedat a particular density. For example, identical barcodes may bepartitioned so that each partition contains about 1, 5, 10, 50, 100,1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000identical barcodes per partition. Barcodes may be partitioned so thateach partition contains at least about 1, 5, 10, 50, 100, 1000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,10000000, 20000000, 50000000, 100000000, or more identical barcodes perpartition. Barcodes may be partitioned so that each partition containsless than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000,500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000,4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000,20000000, 50000000, or 100000000 identical barcodes per partition.Barcodes may be partitioned such that each partition contains about 1-5,5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 identical barcodes perpartition.

Barcodes may be partitioned such that different barcodes are partitionedat a particular density. For example, different barcodes may bepartitioned so that each partition contains about 1, 5, 10, 50, 100,1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000,800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000,7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000different barcodes per partition. Barcodes may be partitioned so thateach partition contains at least about 1, 5, 10, 50, 100, 1000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000,2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,10000000, 20000000, 50000000, 100000000, or more different barcodes perpartition. Barcodes may be partitioned so that each partition containsless than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000,500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000,4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000,20000000, 50000000, or 100000000 different barcodes per partition.Barcodes may be partitioned such that each partition contains about 1-5,5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000,10000-1000000, 10000-10000000, or 10000-100000000 different barcodes perpartition.

The number of partitions employed to partition barcodes may vary, forexample, depending on the application and/or the number of differentbarcodes to be partitioned. For example, the number of partitionsemployed to partition barcodes may be about 5, 10, 50, 100, 250, 500,750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, 20000, 30000, 40000,50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000,500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000,4,000,000, 5,000,000, 10000000, 20000000 or more. The number ofpartitions employed to partition barcodes may be at least about 5, 10,50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000,20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000,300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000,2000000, 3000000, 4000000, 5000000, 10000000, 20000000 or more. Thenumber of partitions employed to partition barcodes may be less thanabout 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500,10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000,200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000,2000000, 3000000, 4000000, 5000000, 10000000, or 20000000. The number ofpartitions employed to partition barcodes may be about 5-10000000,5-5000000, 5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000,1,000-5,000, 1,000-4,000, 1,000-3,000, or 1,000-2,000.

As described above, different barcodes or different sets of barcodes(e.g., each set comprising a plurality of identical barcodes ordifferent barcodes) may be partitioned such that each partitioncomprises a different barcode or different barcode set. In some cases,each partition may comprise a different set of identical barcodes. Wheredifferent sets of identical barcodes are partitioned, the number ofidentical barcodes per partition may vary. For example, about 100,000 ormore different sets of identical barcodes may be partitioned acrossabout 100,000 or more different partitions, such that each partitioncomprises a different set of identical barcodes. In each partition, thenumber of identical barcodes per set of barcodes may be about 1,000,000identical barcodes. In some cases, the number of different sets ofbarcodes may be equal to or substantially equal to the number ofpartitions. Any suitable number of different barcodes or differentbarcode sets (including numbers of different barcodes or differentbarcode sets to be partitioned described elsewhere herein), number ofbarcodes per partition (including numbers of barcodes per partitiondescribed elsewhere herein), and number of partitions (including numbersof partitions described elsewhere herein) may be combined to generate adiverse library of partitioned barcodes with high numbers of barcodesper partition. Thus, as will be appreciated, any of the above-describeddifferent numbers of barcodes may be provided with any of theabove-described barcode densities per partition, and in any of theabove-described numbers of partitions.

The volume of the partitions may vary depending on the application. Forexample, the volume of any of the partitions described in thisdisclosure (e.g., wells, spots, droplets (e.g., in an emulsion), andcapsules) may be about 1000 μl, 900 μl, 800 μl, 700 μl, 600 μl, 500 μl,400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1 μl, 900 nL,800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL,25 nL, 10 nL, 5 nL, 2.5 nL, 1 nL, 900 pL, 800 pL, 700 pL, 600 pL, 500pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 25 pL, 10 pL, 5 pL, 1 pL, 900fL, 800 fL, 700 fL, 600 fL, 500 fL, 400 fL, 300 fL, 200 fL, 100 fL, 50fL, 25 fL, 10 fL, 5 fL, 1 fL, or 0.5 fL. The volume of the partitionsmay be at least about 1000 μl, 900 μl, 800 μl, 700 μl, 600 μl, 500 μl,400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1 μl, 900 nL,800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL,25 nL, 10 nL, 5 nL, 5 nL, 2.5 nL, 1 nL, 900 pL, 800 pL, 700 pL, 600 pL,500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 25 pL, 10 pL, 5 pL, 1 pL,900 fL, 800 fL, 700 fL, 600 fL, 500 fL, 400 fL, 300 fL, 200 fL, 100 fL,50 fL, 25 fL, 10 fL, 5 fL, 1 fL, or 0.5 fL. The volume of the partitionsmay be less than about 1000 μl, 900 μl, 800 μl, 700 μl, 600 μl, 500 μl,400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1 μl, μl, 900nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50nL, 25 nL, 10 nL, 5 nL, 5 nL, 2.5 nL, 1 nL, 900 pL, 800 pL, 700 pL, 600pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 25 pL, 10 pL, 5 pL, 1pL, 900 fL, 800 fL, 700 fL, 600 fL, 500 fL, 400 fL, 300 fL, 200 fL, 100fL, 50 fL, 25 fL, 10 fL, 5 fL, 1 fL, or 0.5 fL. the volume of thepartitions may be about 0.5 fL-5 pL, 10 pL-10 nL, 10 nL-10 μl, 10 μl-100μl, or 100 μl to 1 mL.

There may be variability in the volume of fluid in different partitions.More specifically, the volume of different partitions may vary by atleast (or at most) plus or minus 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or 1000% across aset of partitions. For example, a well (or other partition) may comprisea volume of fluid that is at most 80% of the fluid volume within asecond well (or other partition).

Particular species may also be targeted to specific partitions. Forexample, in some cases, a capture reagent (e.g., an oligonucleotideprobe) may be immobilized or placed within a partition to capturespecific species (e.g., polynucleotides). For example, a captureoligonucleotide may be immobilized on the surface of a bead in order tocapture a species comprising an oligonucleotide with a complementarysequence.

Species may also be partitioned at a particular density. For example,species may be partitioned so that each partition contains about 1, 5,10, 50, 100, 1000, 10000, 100000, or 1000000 species per partition.Species may be partitioned so that each partition contains at leastabout 1, 5, 10, 50, 100, 1000, 10000, 100000, 1000000 or more speciesper partition. Species may be partitioned so that each partitioncontains less than about 1, 5, 10, 50, 100, 1000, 10000, 100000, or1000000 species per partition. Species may be partitioned such that eachpartition contains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000,10000-100000, or 100000-1000000 species per partition.

Species may be partitioned such that at least one partition comprises aspecies that is unique within that partition. This may be true for about1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of thepartitions. This may be true for at least about 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions. This may betrue for less than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90% of the partitions.

a. Wells as Partitions

In some cases, wells are used as partitions. The wells may bemicrowells. A well may comprise a medium comprising a species orplurality of species. Species may be contained within a well in variousconfigurations. In one example, a species is dispensed directly into awell. A species dispensed directly into a well may be overlaid with alayer that is, for example, dissolvable, meltable, or permeable. Thislayer may be, for example, an oil, wax, membrane, or the like. The layermay be dissolved or melted prior to or after introduction of anotherspecies into the well. The well may be sealed at any point, with asealing layer, for example after addition of any species.

In one example, reagents for sample processing are dispensed directlyinto a well and overlaid with a layer that is dissolvable, meltable, orpermeable. A sample comprising an analyte to be processed is introducedon top of the layer. The layer is dissolved or melted, or the analyte(or reagent) diffuses through the layer. The well is sealed andincubated under appropriate conditions for the processing of theanalyte. Processed analyte may then be recovered.

In some cases, wells comprise other partitions. A well may comprise anysuitable partition including, for example, another well, a spot, adroplet (e.g., a droplet in an emulsion), a capsule, a bead, and thelike. Each partition may be present as a single partition or a pluralityof partitions, and each partition may comprise the same species ordifferent species.

In one example, a well comprises a capsule comprising reagents forsample processing. A capsule may be loaded into a well using a liquidmedium, or loaded into a well without a liquid medium (e.g., essentiallydry). As described elsewhere in this disclosure, a capsule may containone or more capsules, or other partitions. A sample comprising ananalyte to be processed may be introduced into the well. The well may besealed and a stimulus may be applied to cause release of the contents ofthe capsule into the well, resulting in contact between the reagents andthe analyte to be processed. The well may be incubated under appropriateconditions for the processing of the analyte. Processed analyte may thenbe recovered. While this example describes an embodiment where a reagentis in a capsule and an analyte is in the well, the oppositeconfiguration—i.e., reagent in the well and analyte in the capsule—isalso possible.

In another example, a well comprises an emulsion and the droplets of theemulsion comprise capsules comprising reagents for sample processing. Asample comprising an analyte to be processed is contained within thedroplets of the emulsion. The well is sealed and a stimulus is appliedto cause release of the contents of the capsules into the droplets,resulting in contact between the reagents and the analyte to beprocessed. The well is incubated under appropriate conditions for theprocessing of the analyte. Processed analyte may then be recovered.While this example describes an embodiment where a reagent is in acapsule and an analyte is in a droplet, the opposite configuration—i.e.,reagent in the droplet and analyte in the capsule—is also possible.

Wells may be arranged as an array, for example a microwell array. Basedon the dimensions of individual wells and the size of the substrate, thewell array may comprise a range of well densities. In some cases, thewell density may be 10 wells/cm², 50 wells/cm², 100 wells/cm², 500wells/cm², 1000 wells/cm², 5000 wells/cm², 10000 wells/cm², 50000wells/cm², or 100000 wells/cm². In some cases, the well density may beat least 10 wells/cm², 50 wells/cm², 100 wells/cm², 500 wells/cm², 1000wells/cm², 5000 wells/cm², 10000 wells/cm², 50000 wells/cm², or 100000wells/cm². In some cases, the well density may be less than 10wells/cm², 50 wells/cm², 100 wells/cm², 500 wells/cm², 1000 wells/cm²,5000 wells/cm², 10000 wells/cm², 50000 wells/cm², or 100000 wells/cm².

b. Spots as Partitions

In some cases, spots are used as partitions. A spot may be made, forexample, by dispensing a substance on a surface. Species may becontained within a spot in various configurations. In one example, aspecies is dispensed directly into a spot by including the species inthe medium used to form the spot. A species dispensed directly onto aspot may be overlaid with a layer that is, for example, dissolvable,meltable, or permeable. This layer may be, for example, an oil, wax,membrane, or the like. The layer may be dissolved or melted prior to orafter introduction of another species onto the spot. The spot may besealed at any point, for example after addition of any species, by anoverlay.

In one example, reagents for sample processing are dispensed directlyonto a spot, for example on a glass slide, and overlaid with a layerthat is dissolvable, meltable, or permeable. A sample comprising ananalyte to be processed is introduced on top of the layer. The layer isdissolved or melted, or the analyte (or reagent) diffuses through thelayer. The spot is sealed and incubated under appropriate conditions forthe processing of the analyte. Processed analyte may then be recovered.

As described elsewhere in this disclosure (e.g., Table 1), spots mayalso be arranged within a well. In some cases, a plurality of spots maybe arranged within a well such that the contents of each spot do notmix. Such a configuration may be useful, for example, when it isdesirable to prevent species from contacting each other. In some cases,a well may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more spots.In some cases, a well may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, or more spots. In some cases, a well may comprise less than 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 spots. In some cases, a well maycomprise 2-4, 2-6, 2-8, 4-6, 4-8, 5-10, or 4-12 spots. Upon addition ofa substance (e.g., a medium containing an analyte) to the well, thespecies in the spot may mix. Moreover, using separate spots to containdifferent species (or combinations of species) may also be useful toprevent cross-contamination of devices used to place the spots insidethe well.

In some cases, spots comprise other partitions. A spot may comprise anysuitable partition including, for example, another spot a droplet (e.g.,a droplet in an emulsion), a capsule, a bead, and the like. Eachpartition may be present as a single partition or a plurality ofpartitions, and each partition may comprise the same species ordifferent species.

In one example, a spot comprises a capsule comprising reagents forsample processing. As described elsewhere in this disclosure, a capsulemay contain one or more capsules, or other partitions. A samplecomprising an analyte to be processed is introduced into the spot. Thespot is sealed and a stimulus is applied to cause release of thecontents of the capsule into the spot, resulting in contact between thereagents and the analyte to be processed. The spot is incubated underappropriate conditions for the processing of the analyte. Processedanalyte may then be recovered. While this example describes anembodiment where a reagent is in a capsule and an analyte is in thespot, the opposite configuration—i.e., reagent in the spot and analytein the capsule—is also possible.

In another example, a spot comprises an emulsion and the droplets of theemulsion comprise capsules comprising reagents for sample processing. Asample comprising an analyte to be processed is contained within thedroplets of the emulsion. The spot is sealed and a stimulus is appliedto cause release of the contents of the capsules into the droplets,resulting in contact between the reagents and the analyte to beprocessed. The spot is incubated under appropriate conditions for theprocessing of the analyte. Processed analyte may then be recovered.While this example describes an embodiment where a reagent is in acapsule and an analyte is in a droplet, the opposite configuration—i.e.,reagent in the droplet and analyte in the capsule—is also possible.

Spots may be of uniform size or heterogeneous size. In some cases, thediameter of a spot may be about 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800μm, 900 μm, 1 mm, 2 mm, 5 mm, or 1 cm. A spot may have a diameter of atleast about 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm,200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 1mm, 2 mm, 5 mm, or 1 cm. In some cases, a spot may have a diameter ofless than about 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1mm, 1 mm, 2 mm, 5 mm, or 1 cm. In some cases, a spot may have a diameterof about 0.1 μm to 1 cm, 100 μm to 1 mm, 100 μm to 500 μm, 100 μm to 600μm, 150 μm to 300 μm, or 150 μm to 400 μm.

Spots may be arranged as an array, for example a spot array. Based onthe dimensions of individual spots and the size of the substrate, thespot array may comprise a range of spot densities. In some cases, thespot density may be 10 spots/cm², 50 spots/cm², 100 spots/cm², 500spots/cm², 1000 spots/cm², 5000 spots/cm², 10000 spots/cm², 50000spots/cm², or 100000 spots/cm². In some cases, the spot density may beat least 10 spots/cm², 50 spots/cm², 100 spots/cm², 500 spots/cm², 1000spots/cm², 5000 spots/cm², 10000 spots/cm², 50000 spots/cm², or 100000spots/cm². In some cases, the spot density may be less than 10spots/cm², 50 spots/cm², 100 spots/cm², 500 spots/cm², 1000 spots/cm²,5000 spots/cm², 10000 spots/cm², 50000 spots/cm², or 100000 spots/cm².

c. Emulsions as Partitions

In some cases, the droplets in an emulsion are used as partitions. Anemulsion may be prepared, for example, by any suitable method, includingmethods known in the art. (See e.g., Weizmann et al., Nature Methods,2006, 3(7):545-550; Weitz et al. U.S. Pub. No. 2012/0211084). In somecases, water-in-fluorocarbon emulsions may be used. These emulsions mayincorporate fluorosurfactants such as oligomeric perfluorinatedpolyethers (PFPE) with polyethylene glycol (PEG). (Holtze et al., Lab ona Chip, 2008, 8(10):1632-1639). In some cases, monodisperse emulsionsmay be formed in a microfluidic flow focusing device. (Garstecki et al.,Applied Physics Letters, 2004, 85(13):2649-2651). The droplet maycomprise, for example, one or more reagents (e.g., restriction enzymes,ligases, polymerases, reagents necessary for nucleic acid amplification(e.g., primers, DNA polymerases, dNTPs, buffers)), a polynucleotidesample, and a barcode sequence. In some cases, the barcode sequence,polynucleotide sample, or any reagent may be associated with a solidsurface within a droplet. In some cases, the solid surface is a bead. Insome cases, the bead is a gel bead (see e.g., Agresti et al., U.S.Patent Publication No. 2010/0136544). In some cases the droplet ishardened into a gel bead (e.g., via polymerization).

A species may be contained within a droplet in an emulsion containing,for example, a first phase (e.g., oil or water) forming the droplet anda second (continuous) phase (e.g., water or oil). An emulsion may be asingle emulsion, for example, a water-in-oil or an oil-in-wateremulsion. An emulsion may be a double emulsion, for example awater-in-oil-in-water or an oil-in-water-in-oil emulsion. Higher-orderemulsions are also possible. The emulsion may be held in any suitablecontainer, including any suitable partition described in thisdisclosure.

In some cases, droplets in an emulsion comprise other partitions. Adroplet in an emulsion may comprise any suitable partition including,for example, another droplet (e.g., a droplet in an emulsion), acapsule, a bead, and the like. Each partition may be present as a singlepartition or a plurality of partitions, and each partition may comprisethe same species or different species.

In one example, a droplet in an emulsion comprises a capsule comprisingreagents for sample processing. As described elsewhere in thisdisclosure, a capsule may contain one or more capsules, or otherpartitions. A sample comprising an analyte to be processed is containedwithin the droplet. A stimulus is applied to cause release of thecontents of the capsule into the droplet, resulting in contact betweenthe reagents and the analyte to be processed. The droplet is incubatedunder appropriate conditions for the processing of the analyte.Processed analyte may then be recovered. While this example describes anembodiment where a reagent is in a capsule and an analyte is in thedroplet, the opposite configuration—i.e., reagent in the droplet andanalyte in the capsule—is also possible.

The droplets in an emulsion may be of uniform size or heterogeneoussize. In some cases, the diameter of a droplet in an emulsion may beabout 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800μm, 900 μm, or 1 mm. A droplet may have a diameter of at least about0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm,100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm,900 μm, or 1 mm. In some cases, a droplet may have a diameter of lessthan about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700μm, 800 μm, 900 μm, or 1 mm. In some cases, a droplet may have adiameter of about 0.001 μm to 1 mm, 0.01 μm to 900 μm, 0.1 μm to 600 μm,100 μm to 200 μm, 100 μm to 300 μm, 100 μm to 400 μm, 100 μm to 500 μm,100 μm to 600 μm, 150 μm to 200 μm, 150 μm to 300 μm, or 150 μm to 400μm.

Droplets in an emulsion also may have a particular density. In somecases, the droplets are less dense than an aqueous fluid (e.g., water);in some cases, the droplets are denser than an aqueous fluid. In somecases, the droplets are less dense than a non-aqueous fluid (e.g., oil);in some cases, the droplets are denser than a non-aqueous fluid.Droplets may have a density of about 0.05 g/cm³, 0.1 g/cm³, 0.2 g/cm³,0.3 g/cm³, 0.4 g/cm³, 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³, 0.8 g/cm³, 0.81g/cm³, 0.82 g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87g/cm³, 0.88 g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93g/cm³, 0.94 g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99g/cm³, 1.00 g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³,2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. Droplets mayhave a density of at least about 0.05 g/cm³, 0.1 g/cm³, 0.2 g/cm³, 0.3g/cm³, 0.4 g/cm³, 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³, 0.8 g/cm³, 0.81g/cm³, 0.82 g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87g/cm³, 0.88 g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93g/cm³, 0.94 g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99g/cm³, 1.00 g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³,2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. In othercases, droplet densities may be at most about 0.7 g/cm³, 0.8 g/cm³, 0.81g/cm³, 0.82 g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87g/cm³, 0.88 g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93g/cm³, 0.94 g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99g/cm³, 1.00 g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³,2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. Such densitiescan reflect the density of the capsule in any particular fluid (e.g.,aqueous, water, oil, etc.)

d. Capsules as Partitions

In some cases, capsules are used as partitions. A capsule may beprepared by any suitable method, including methods known in the art,including emulsification polymerization (Weitz et al. (U.S. Pub. No.2012/0211084)), layer-by-layer assembly with polyelectrolytes,coacervation, internal phase separation, and flow focusing. Any suitablespecies may be contained within a capsule. The capsule may be held inany suitable container, including any suitable partition described inthis disclosure.

In some cases, capsules comprise other partitions. A capsule maycomprise any suitable partition including, for example, another capsule,a droplet in an emulsion, a bead, and the like. Each partition may bepresent as a single partition or a plurality of partitions, and eachpartition may comprise the same species or different species.

In one example, an outer capsule comprises an inner capsule. The innercapsule comprises reagents for sample processing. An analyte isencapsulated in the medium between the inner capsule and the outercapsule. A stimulus is applied to cause release of the contents of theinner capsule into the outer capsule, resulting in contact between thereagents and the analyte to be processed. The outer capsule is incubatedunder appropriate conditions for the processing of the analyte.Processed analyte may then be recovered. While this example describes anembodiment where a reagent is in an inner capsule and an analyte in themedium between the inner capsule and the outer capsule, the oppositeconfiguration—i.e., reagent in the medium between the inner capsule andthe outer capsule, and analyte in the inner capsule—is also possible.

Capsules may be pre-formed and filled with reagents by injection. Forexample, the picoinjection methods described in Abate et al. (Proc.Natl. Acad. Sci. U.S.A., 2010, 107(45), 19163-19166) and Weitz et al.(U.S. Pub. No. 2012/0132288) may be used to introduce reagents into theinterior of capsules described herein. Generally, the picoinjection willbe performed prior to the hardening of the capsule shell, for example byinjecting species into the interior of a capsule precursor, such as adroplet of an emulsion, before formation of the capsule shell.

Capsules may be of uniform size or heterogeneous size. In some cases,the diameter of a capsule may be about 0.001 μm, 0.01 μm, 0.05 μm, 0.1μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm,400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. A capsule mayhave a diameter of at least about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm,0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. In some cases, acapsule may have a diameter of less than about 0.001 μm, 0.01 μm, 0.05μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm,300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. In somecases, a capsule may have a diameter of about 0.001 μm to 1 mm, 0.01 μmto 900 μm, 0.1 μm to 600 μm, 100 μm to 200 μm, 100 μm to 300 μm, 100 μmto 400 μm, 100 μm to 500 μm, 100 μm to 600 μm, 150 μm to 200 μm, 150 μmto 300 μm, or 150 μm to 400 μm.

Capsules also may have a particular density. In some cases, the capsulesare less dense than an aqueous fluid (e.g., water); in some cases, thecapsules are denser than an aqueous fluid. In some cases, the capsulesare less dense than a non-aqueous fluid (e.g., oil); in some cases, thecapsules are denser than a non-aqueous fluid. Capsules may have adensity of about 0.05 g/cm³, 0.1 g/cm³, 0.2 g/cm³, 0.3 g/cm³, 0.4 g/cm³,0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³, 0.8 g/cm³, 0.81 g/cm³, 0.82 g/cm³, 0.83g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87 g/cm³, 0.88 g/cm³, 0.89g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93 g/cm³, 0.94 g/cm³, 0.95g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99 g/cm³, 1.00 g/cm³, 1.05g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4 g/cm³, 1.5 g/cm³, 1.6 g/cm³,1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³, 2.1 g/cm³, 2.2 g/cm³, 2.3g/cm³, 2.4 g/cm³, or 2.5 g/cm³. Capsules may have a density of at leastabout 0.05 g/cm³, 0.1 g/cm³, 0.2 g/cm³, 0.3 g/cm³, 0.4 g/cm³, 0.5 g/cm³,0.6 g/cm³, 0.7 g/cm³, 0.8 g/cm³, 0.81 g/cm³, 0.82 g/cm³, 0.83 g/cm³,0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87 g/cm³, 0.88 g/cm³, 0.89 g/cm³,0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93 g/cm³, 0.94 g/cm³, 0.95 g/cm³,0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99 g/cm³, 1.00 g/cm³, 1.05 g/cm³,1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4 g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³, 2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³,2.4 g/cm³, or 2.5 g/cm³. In other cases, capsule densities may be atmost about 0.7 g/cm³, 0.8 g/cm³, 0.81 g/cm³, 0.82 g/cm³, 0.83 g/cm³,0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87 g/cm³, 0.88 g/cm³, 0.89 g/cm³,0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93 g/cm³, 0.94 g/cm³, 0.95 g/cm³,0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99 g/cm³, 1.00 g/cm³, 1.05 g/cm³,1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4 g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³, 2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³,2.4 g/cm³, or 2.5 g/cm³. Such densities can reflect the density of thecapsule in any particular fluid (e.g., aqueous, water, oil, etc.)

1. Production of Capsules by Flow Focusing

In some cases, capsules may be produced by flow focusing. Flow focusingis a method whereby a first fluid that is immiscible with a second fluidis flowed into the second fluid. With reference to FIG. 12, a first(e.g., aqueous) fluid comprising a monomer, crosslinker, initiator, andaqueous surfactant 1201 is flowed into a second (e.g., oil) fluidcomprising a surfactant and an accelerator 1202. After entering thesecond fluid at a T-junction in a microfluidic device 1203, a droplet offirst fluid breaks off from the first fluid stream and a capsule shellbegins to form 1204 due to the mixing of the monomer, crosslinker, andinitiator in the first fluid and the accelerator in the second fluid.Thus, a capsule is formed. As the capsule proceeds downstream, the shellbecomes thicker due to increased exposure to the accelerator. Varyingthe concentrations of the reagents may also be used to vary thethickness and permeability of the capsule shell.

A species, or other partition such as a droplet, may be encapsulated by,for example, including the species in the first fluid. Including thespecies in the second fluid may embed the species in the shell of thecapsule. Of course, depending on the needs of the particular sampleprocessing method, the phases may also be reversed—i.e., the first phasemay be an oil phase and the second phase may be an aqueous phase.

2. Production of Capsules within Capsules by Flow Focusing

In some cases, capsules within capsules may be produced by flowfocusing. With reference to FIG. 13, a first (e.g., aqueous) fluidcomprising a capsule, monomer, crosslinker, initiator, and aqueoussurfactant 1301 is flowed into a second (oil) fluid comprising asurfactant and an accelerator 1302. After entering the second fluid at aT-junction in a microfluidic device 1303, a droplet of first fluidbreaks off from the first fluid stream and a second capsule shell beginsto form around the capsule 1304 due to the mixing of the monomer,crosslinker, and initiator in the first fluid and the accelerator in thesecond fluid. Thus, a capsule within a capsule is formed. As the capsuleproceeds downstream, the shell becomes thicker due to increased exposureto the accelerator. Varying the concentrations of the reagents may alsobe used to vary the thickness and permeability of the second capsuleshell.

A species may be encapsulated by, for example, including the species inthe first fluid. Including the species in the second fluid may embed thespecies in the second shell of the capsule. Of course, depending on theneeds of the particular sample processing method, the phases may also bereversed—i.e., the first phase may be an oil phase and the second phasemay be an aqueous phase.

3. Production of Capsules in Batch

In some cases, capsules may be produced in batch, using capsuleprecursors, such as the droplets in an emulsion. Capsule precursors maybe formed by any suitable method, for example by producing an emulsionwith droplets comprising a monomer, a crosslinker, an initiator, and asurfactant. An accelerator may then be added to the medium, resulting inthe formation of capsules. As for the methods of flow focusing, thethickness of the shell can be varied by varying the concentrations ofthe reactants, and the time of exposure to the accelerator. The capsulesmay then be washed and recovered. As for any method described herein, aspecies, including other partitions, may be encapsulated within thecapsule or, if suitable, within the shell.

In another example, the droplets of an emulsion may be exposed to anaccelerator that is present in an outlet well during the emulsiongeneration process. For example, capsule precursors may be formed by anysuitable method, such as the flow focusing method illustrated in FIG.12. Rather than including the accelerator in second fluid 1202, theaccelerator may be included in a medium located at the exit of theT-junction (e.g., a medium located at the far-right of the horizontalchannel of FIG. 12. As the emulsion droplets (i.e., capsule precursors)exit the channel, they contact the medium comprising the accelerator(i.e., the outlet medium). If the capsule precursor has a density thatis less than the density of outlet medium, the capsule precursors willrise through the medium, ensuring convectional and diffusional exposureto the accelerator and reducing the likelihood of polymerization at theoutlet of the channel.

VI. Species

The methods, compositions, systems, devices, and kits of this disclosuremay be used with any suitable species. A species can be, for example,any substance used in sample processing, such as a reagent or ananalyte. Exemplary species include whole cells, chromosomes,polynucleotides, organic molecules, proteins, polypeptides,carbohydrates, saccharides, sugars, lipids, enzymes, restrictionenzymes, ligases, polymerases, barcodes, adapters, small molecules,antibodies, fluorophores, deoxynucleotide triphosphates (dNTPs),dideoxynucleotide triphosphates (ddNTPs), buffers, acidic solutions,basic solutions, temperature-sensitive enzymes, pH-sensitive enzymes,light-sensitive enzymes, metals, metal ions, magnesium chloride, sodiumchloride, manganese, aqueous buffer, mild buffer, ionic buffer,inhibitors, saccharides, oils, salts, ions, detergents, ionicdetergents, non-ionic detergents, oligonucleotides, nucleotides, DNA,RNA, peptide polynucleotides, complementary DNA (cDNA), double strandedDNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA,chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA(mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA,scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA, a lockednucleic acid (LNA) in whole or part, locked nucleic acid nucleotides,any other type of nucleic acid analogue, proteases, nucleases, proteaseinhibitors, nuclease inhibitors, chelating agents, reducing agents,oxidizing agents, probes, chromophores, dyes, organics, emulsifiers,surfactants, stabilizers, polymers, water, small molecules,pharmaceuticals, radioactive molecules, preservatives, antibiotics,aptamers, and the like. In summary, the species that are used will varydepending on the particular sample processing needs.

In some cases, a partition comprises a set of species that have asimilar attribute (e.g., a set of enzymes, a set of minerals, a set ofoligonucleotides, a mixture of different barcodes, a mixture ofidentical barcodes). In other cases, a partition comprises aheterogeneous mixture of species. In some cases, the heterogeneousmixture of species comprises all components necessary to perform aparticular reaction. In some cases, such mixture comprises allcomponents necessary to perform a reaction, except for 1, 2, 3, 4, 5, ormore components necessary to perform the reaction. In some cases, suchadditional components are contained within a different partition orwithin a solution within or surrounding a partition.

A species may be naturally-occurring or synthetic. A species may bepresent in a sample obtained using any methods known in the art. In somecases, a sample may be processed before analyzing it for an analyte.

A species may be obtained from any suitable location, including fromorganisms, whole cells, cell preparations and cell-free compositionsfrom any organism, tissue, cell, or environment. A species may beobtained from environmental samples, biopsies, aspirates, formalin fixedembedded tissues, air, agricultural samples, soil samples, petroleumsamples, water samples, or dust samples. In some instances, a speciesmay be obtained from bodily fluids which may include blood, urine,feces, serum, lymph, saliva, mucosal secretions, perspiration, centralnervous system fluid, vaginal fluid, or semen. Species may also beobtained from manufactured products, such as cosmetics, foods, personalcare products, and the like. Species may be the products of experimentalmanipulation including, recombinant cloning, polynucleotideamplification, polymerase chain reaction (PCR) amplification,purification methods (such as purification of genomic DNA or RNA), andsynthesis reactions.

In some cases, a species may quantified by mass. A species may beprovided in a mass of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,9500, 10000 ng, 1 μg, 5 μg, 10 μg, 15 μg, or 20 μg. A species may beprovided in a mass of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, 9500, 10000 ng, 1 μg, 5 μg, 10 μg, 15 μg, or 20 μg. Aspecies may be provided in a mass of less than about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000,7500, 8000, 8500, 9000, 9500, 10000 ng, 1 μg, 5 μg, 10 μg, 15 μg, or 20μg. A species may be provided in a mass ranging from about 1-10, 10-50,50-100, 100-200, 200-1000, 1000-10000 ng, 1-5 μg, or 1-20 μg. Asdescribed elsewhere in this disclosure, if a species is apolynucleotide, amplification may be used to increase the quantity of apolynucleotide.

Polynucleotides may also be quantified as “genome equivalents.” A genomeequivalent is an amount of polynucleotide equivalent to one haploidgenome of an organism from which the target polynucleotide is derived.For example, a single diploid cell contains two genome equivalents ofDNA. Polynucleotides may be provided in an amount ranging from about1-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, or100000-1000000 genome equivalents. Polynucleotides may be provided in anamount of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,9500, 10000, 20000, 30000, 40000, 50000, 60000 70000, 80000, 90000,100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000,or 1000000 genome equivalents. Polynucleotides may be provided in anamount less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,10000, 20000, 30000, 40000, 50000, 60000 70000, 80000, 90000, 100000,200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or1000000 genome equivalents.

Polynucleotides may also be quantified by the amount of sequencecoverage provided. The amount of sequence coverage refers to the averagenumber of reads representing a given nucleotide in a reconstructedsequence. Generally, the greater the number of times a region issequenced, the more accurate the sequence information obtained.Polynucleotides may be provided in an amount that provides a range ofsequence coverage from about 0.1×-10×, 10×-50×, 50×-100×, 100×-200×, or200×-500×. Polynucleotides may be provided in an amount that provides atleast about 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×,5×, 10×, 25×, 50×, 100×, 125×, 150×, 175×, or 200× sequence coverage.Polynucleotides may be provided in an amount that provides less thanabout 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 5×, 10×,25×, 50×, 100×, 125×, 150×, 175×, or 200× sequence coverage.

In some cases, species are introduced into a partition either before orafter a particular step. For example, a lysis buffer reagent may beintroduced into a partition following partitioning of a cellular sampleinto the partitions. In some cases, reagents and/or partitionscomprising reagents are introduced sequentially such that differentreactions or operations occur at different steps. The reagents (orpartitions comprising reagents) may be also be loaded at stepsinterspersed with a reaction or operation step. For example, capsulescomprising reagents for fragmenting molecules (e.g., nucleic acids) maybe loaded into a well, followed by a fragmentation step, which may befollowed by loading of capsules comprising reagents for ligatingbarcodes (or other unique identifiers, e.g., antibodies) and subsequentligation of the barcodes to the fragmented molecules.

VII. Processing of Analytes and Other Species

In some cases, the methods, compositions, systems, devices, and kits ofthis disclosure may be used to process a sample containing a species,for example an analyte. Any suitable process can be performed.

a. Preparation of Target Polynucleotides

Target polynucleotides processed according to the methods provided inthis disclosure may be DNA, RNA, peptide nucleic acids, and any hybridthereof, where the polynucleotide contains any combination of deoxyribo-and ribo-nucleotides. Polynucleotides may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. Polynucleotides may contain any combination ofnucleotides, including uracil, adenine, thymine, cytosine, guanine,inosine, xanthine, hypoxanthine, isocytosine, isoguanine and anynucleotide derivative thereof. As used herein, the term “nucleotide” mayinclude nucleotides and nucleosides, as well as nucleoside andnucleotide analogs, and modified nucleotides, including both syntheticand naturally occurring species. Target polynucleotides may be cDNA,mitochondrial DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA),transfer RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajalbody-specific RNA (scaRNA), microRNA (miRNA), double stranded (dsRNA),ribozyme, riboswitch or viral RNA. Target polynucleotides may becontained on a plasmid, cosmid, or chromosome, and may be part of agenome. In some cases, a target polynucleotide may comprise one or moregenes and/or one or more pseudogenes. A pseudogene generally refers to adysfunctional relative of a gene that has lost its protein codingability and/or is otherwise no longer expressed in the cell.

Target polynucleotides may be obtained from a sample using any methodsknown in the art. A target polynucleotide processed as described hereinmay be obtained from whole cells, cell preparations and cell-freecompositions from any organism, tissue, cell, or environment. In someinstances, target polynucleotides may be obtained from bodily fluidswhich may include blood, urine, serum, lymph, saliva, mucosalsecretions, perspiration, or semen. In some instances, polynucleotidesmay be obtained from environmental samples including air, agriculturalproducts, water, and soil. In other instances polynucleotides may be theproducts of experimental manipulation including, recombinant cloning,polynucleotide amplification (as generally described in PCT/US99/01705),polymerase chain reaction (PCR) amplification, purification methods(such as purification of genomic DNA or RNA), and synthesis reactions.

Genomic DNA may be obtained from naturally occurring or geneticallymodified organisms or from artificially or synthetically createdgenomes. Target polynucleotides comprising genomic DNA may be obtainedfrom any source and using any methods known in the art. For example,genomic DNA may be isolated with or without amplification. Amplificationmay include PCR amplification, multiple displacement amplification(MDA), rolling circle amplification and other amplification methods.Genomic DNA may also be obtained by cloning or recombinant methods, suchas those involving plasmids and artificial chromosomes or otherconventional methods (see Sambrook and Russell, Molecular Cloning: ALaboratory Manual., cited supra.) Polynucleotides may be isolated usingother methods known in the art, for example as disclosed in GenomeAnalysis: A Laboratory Manual Series (Vols. I-IV) or Molecular Cloning:A Laboratory Manual. If the isolated polynucleotide is an mRNA, it maybe reverse transcribed into cDNA using conventional techniques, asdescribed in Sambrook and Russell, Molecular Cloning: A LaboratoryManual., cited supra.

Target polynucleotides may also be isolated from “target organisms” or“target cells”. The terms “target organism” and “target cell” refer toan organism or cell, respectively, from which target polynucleotides maybe obtained. Target cells may be obtained from a variety of organismsincluding human, mammal, non-human mammal, ape, monkey, chimpanzee,plant, reptilian, amphibian, avian, fungal, viral or bacterialorganisms. Target cells may also be obtained from a variety of clinicalsources such as biopsies, aspirates, blood, urine, formalin fixedembedded tissues, and the like. Target cells may comprise a specificcell type, such as a somatic cell, germline cell, wild-type cell, canceror tumor cells, or diseased or infected cell. A target cell may refer toa cell derived from a particular tissue or a particular locus in atarget organism A target cell may comprise whole intact cells, or cellpreparations.

Target polynucleotides may also be obtained or provided in specifiedquantities. Amplification may be used to increase the quantity of atarget polynucleotide. Target polynucleotides may quantified by mass.For example, target polynucleotides may be provided in a mass rangingfrom about 1-10, 10-50, 50-100, 100-200, 200-1000, 1000-10000 ng. Targetpolynucleotides may be provided in a mass of at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,7000, 7500, 8000, 8500, 9000, 9500, or 10000 ng. Target polynucleotidesmay be provided in a mass of less than about 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, 9500, or 10000 ng.

Target polynucleotides may also be quantified as “genome equivalents.” Agenome equivalent is an amount of polynucleotide equivalent to onehaploid genome of an organism from which the target polynucleotide isderived. For example, a single diploid cell contains two genomeequivalents of DNA. Target polynucleotides may be provided in an amountranging from about 1-10, 10-50, 50-100, 100-1000, 1000-10000,10000-100000, or 100000-1000000 genome equivalents. Targetpolynucleotides may be provided in an amount of at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100,500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000,6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000, 40000,50000, 60000 70000, 80000, 90000, 100000, 200000, 300000, 400000,500000, 600000, 700000, 800000, 900000, or 1000000 genome equivalents.Target polynucleotides may be provided in an amount less than about 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50,100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000,40000, 50000, 60000 70000, 80000, 90000, 100000, 200000, 300000, 400000,500000, 600000, 700000, 800000, 900000, or 1000000 genome equivalents.

Target polynucleotide may also be quantified by the amount of sequencecoverage provided. The amount of sequence coverage refers to the averagenumber of reads representing a given nucleotide in a reconstructedsequence. Generally, the greater the number of times a region issequenced, the more accurate the sequence information obtained. Targetpolynucleotides may be provided in an amount that provides a range ofsequence coverage from about 0.1×-10×, 10-×-50×, 50×-100×, 100×-200×, or200×-500×. Target polynucleotide may be provided in an amount thatprovides at least about 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×,0.9×, 1.0×, 5×, 10×, 25×, 50×, 100×, 125×, 150×, 175×, or 200× sequencecoverage. Target polynucleotide may be provided in an amount thatprovides less than about 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×,1.0×, 5×, 10×, 25×, 50×, 100×, 125×, 150×, 175×, or 200× sequencecoverage.

b. Fragmentation of Target Polynucleotides

In some cases, the methods, compositions, systems, devices, and kits ofthis disclosure may be used for polynucleotide fragmentation.Fragmentation of polynucleotides is used as a step in a variety ofmethods, including polynucleotide sequencing. The size of thepolynucleotide fragments, typically described in terms of length(quantified by the linear number of nucleotides per fragment), may varydepending on the source of the target polynucleotide, the method usedfor fragmentation, and the desired application. A single fragmentationstep or a plurality of fragmentation steps may be used.

Fragments generated using the methods described herein may be about1-10, 10-20, 20-50, 50-100, 50-200, 100-200, 200-300, 300-400, 400-500,500-1000, 1000-5000, 5000-10000, 10000-100000, 100000-250000, or250000-500000 nucleotides in length. Fragments generated using themethods described herein may be at least about 10, 20, 100, 200, 300,400, 500, 1000, 5000, 10000, 100000, 250000, 500000, or more nucleotidesin length. Fragments generated using the methods described herein may beless than about 10, 20, 100, 200, 300, 400, 500, 1000, 5000, 10000,100000, 250000, 500000, nucleotides in length.

Fragments generated using the methods described herein may have a meanor median length of about 1-10, 10-20, 20-50, 50-100, 50-200, 100-200,200-300, 300-400, 400-500, 500-1000, 1000-5000, 5000-10000,10000-100000, 100000-250000, or 250000-500000 nucleotides. Fragmentsgenerated using the methods described herein may have a mean or medianlength of at least about 10, 20, 100, 200, 300, 400, 500, 1000, 5000,10000, 100000, 250000, 500000, or more nucleotides. Fragments generatedusing the methods described herein may have a mean or median length ofless than about 10, 20, 100, 200, 300, 400, 500, 1000, 5000, 10000,100000, 250000, 500000, nucleotides.

Numerous fragmentation methods are known in the art. For example,fragmentation may be performed through physical, mechanical or enzymaticmethods. Physical fragmentation may include exposing a targetpolynucleotide to heat or to UV light. Mechanical disruption may be usedto mechanically shear a target polynucleotide into fragments of thedesired range. Mechanical shearing may be accomplished through a numberof methods known in the art, including repetitive pipetting of thetarget polynucleotide, sonication (e.g., using ultrasonic waves),cavitation and nebulization. Target polynucleotides may also befragmented using enzymatic methods. In some cases, enzymatic digestionmay be performed using enzymes such as using restriction enzymes.

While the methods of fragmentation described in the preceding paragraph,and in some paragraphs of the disclosure, are described with referenceto “target” polynucleotides, this is not meant to be limiting, above oranywhere else in this disclosure. Any method of fragmentation describedherein, or known in the art, can be applied to any polynucleotide usedwith the invention. In some cases, this polynucleotide may be a targetpolynucleotide, such as a genome. In other cases, this polynucleotidemay be a fragment of a target polynucleotide which one wishes to furtherfragment. In still other cases, still further fragments may be stillfurther fragmented. Any suitable polynucleotide may be fragmentedaccording the methods described herein.

A fragment of a polynucleotide generally comprises a portion of thesequence of the targeted polynucleotide from which the fragment wasgenerated. In some cases, a fragment may comprise a copy of a geneand/or pseudogene, including one included in the original targetpolynucleotide. In some cases, a plurality of fragments generated fromfragmenting a target polynucleotide may comprise fragments that eachcomprise a copy of a gene and/or pseudogene.

Restriction enzymes may be used to perform specific or non-specificfragmentation of target polynucleotides. The methods of the presentdisclosure may use one or more types of restriction enzymes, generallydescribed as Type I enzymes, Type II enzymes, and/or Type III enzymes.Type II and Type III enzymes are generally commercially available andwell known in the art. Type II and Type III enzymes recognize specificsequences of nucleotide base pairs within a double strandedpolynucleotide sequence (a “recognition sequence” or “recognitionsite”). Upon binding and recognition of these sequences, Type II andType III enzymes cleave the polynucleotide sequence. In some cases,cleavage will result in a polynucleotide fragment with a portion ofoverhanging single stranded DNA, called a “sticky end.” In other cases,cleavage will not result in a fragment with an overhang, creating a“blunt end.” The methods of the present disclosure may comprise use ofrestriction enzymes that generate either sticky ends or blunt ends.

Restriction enzymes may recognize a variety of recognition sites in thetarget polynucleotide. Some restriction enzymes (“exact cutters”)recognize only a single recognition site (e.g., GAATTC). Otherrestriction enzymes are more promiscuous, and recognize more than onerecognition site, or a variety of recognition sites. Some enzymes cut ata single position within the recognition site, while others may cut atmultiple positions. Some enzymes cut at the same position within therecognition site, while others cut at variable positions.

The present disclosure provides method of selecting one or morerestriction enzymes to produce fragments of a desired length.Polynucleotide fragmentation may be simulated in silico, and thefragmentation may be optimized to obtain the greatest number or fractionof polynucleotide fragments within a particular size range, whileminimizing the number or fraction of fragments within undesirable sizeranges. Optimization algorithms may be applied to select a combinationof two or more enzymes to produce the desired fragment sizes with thedesired distribution of fragments quantities.

A polynucleotide may be exposed to two or more restriction enzymessimultaneously or sequentially. This may be accomplished by, forexample, adding more than one restriction enzyme to a partition, or byadding one restriction enzyme to a partition, performing the digestion,deactivating the restriction enzyme (e.g., by heat treatment) and thenadding a second restriction enzyme. Any suitable restriction enzyme maybe used alone, or in combination, in the methods presented herein.

In some cases, a species is a restriction enzyme that is a“rare-cutter.” The term “rare-cutter enzyme,” as used herein, generallyrefers to an enzyme with a recognition site that occurs only rarely in agenome. The size of restriction fragments generated by cutting ahypothetical random genome with a restriction enzyme may be approximatedby 4^(N), where N is the number of nucleotides in the recognition siteof the enzyme. For example, an enzyme with a recognition site consistingof 7 nucleotides would cut a genome once every 4⁷ bp, producingfragments of about 16,384 bp. Generally rare-cutter enzymes haverecognition sites comprising 6 or more nucleotides. For example, a rarecutter enzyme may have a recognition site comprising or consisting of 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. Examples of rare-cutterenzymes include NotI (GCGGCCGC), XmaIII (CGGCCG), SstII (CCGCGG), SalI(GTCGAC), NruI (TCGCGA), NheI (GCTAGC), Nb.BbvCI (CCTCAGC), BbvCI(CCTCAGC), AscI (GGCGCGCC), AsiSI (GCGATCGC), FseI (GGCCGGCC), PacI(TTAATTAA), PmeI (GTTTAAAC), SbfI (CCTGCAGG), SgrAI (CRCCGGYG), SwaI(ATTTAAAT), BspQI (GCTCTTC), SapI (GCTCTTC), SfiI (GGCCNNNNNGGCC) (SEQID NO: 27), CspCI (CAANNNNNGTGG) (SEQ ID NO: 28), AbsI (CCTCGAGG), CciNI(GCGGCCGC), FspAI (RTGCGCAY), MauBI (CGCGCGCG), MreI (CGCCGGCG), MssI(GTTTAAAC), PalAI (GGCGCGCC), RgaI (GCGATCGC), RigI (GGCCGGCC), SdaI(CCTGCAGG), SfaAI (GCGATCGC), SgfI (GCGATCGC), SgrDI (CGTCGACG), SgsI(GGCGCGCC), SmiI (ATTTAAAT), SrfI (GCCCGGGC), Sse2321 (CGCCGGCG),Sse83871 (CCTGCAGG), LguI (GCTCTTC), PciSI (GCTCTTC), AarI (CACCTGC),AjuI (GAANNNNNNNTTGG) (SEQ ID NO: 29), AloI (GAACNNNNNNTCC) (SEQ ID NO:30), BarI (GAAGNNNNNNTAC) (SEQ ID NO: 31), PpiI (GAACNNNNNCTC) (SEQ IDNO: 32), PsrI (GAACNNNNNNTAC) (SEQ ID NO: 33), and others.

In some cases, polynucleotides may be fragmented and barcoded at thesame time. For example, a transposase (e.g., NEXTERA) may be used tofragment a polynucleotide and add a barcode to the polynucleotide.

Fragmenting of a target polynucleotide may occur prior to partitioningof the target polynucleotide or fragments generated from fragmenting.For example, genomic DNA (gDNA) may be fragmented, using, for example, arestriction enzyme, prior to the partitioning of its generatedfragments. In another example, a target polynucleotide may be enteredinto a partition along with reagents necessary for fragmentation (e.g.,including a restriction enzyme), such that fragmentation of the targetpolynucleotide occurs within the partition. For example, gDNA may befragmented in a partition comprising a restriction enzyme, and therestriction enzyme is used to fragment the gDNA.

In some cases, a plurality of fragments may be generated prior topartitioning, using any method for fragmentation described herein. Someor all of the fragments of the plurality, for example, may each comprisea copy of a gene and/or a pseudogene. The fragments can be separated andpartitioned such that each copy of the gene or pseudogene is located ina different partition. Each partition, for example, can comprise adifferent barcode sequence such that each copy of the gene and/orpseudogene can be associated with a different barcode sequence, usingbarcoding methods described elsewhere herein. Via the different barcodesequences, each gene and/or pseudogene can be counted and/ordifferentiated during sequencing of the barcoded fragments. Anysequencing method may be used, including those described herein.

For example, using restriction enzymes, genomic DNA (gDNA) can befragmented to generate a plurality of non-overlapping fragments of thegDNA. At least some of the fragments of the plurality may each comprisea copy of a gene and/or a pseudogene. The fragments may be separated andpartitioned such that each copy of the gene or pseudogene is located ina different partition. Each partition, for example, can comprise adifferent barcode sequence such that each copy of the gene and/orpseudogene may be barcoded with a different barcode sequence. Via thedifferent barcode sequences, the genes and/or pseudogenes may be countedand or differentiated after sequencing of the barcoded fragments. Anysequencing method may be used, including those described herein.

VIII. Stimuli-Responsiveness

In some cases, stimuli may be used to trigger the release of a speciesfrom a partition. Generally, a stimulus may cause disruption of thestructure of a partition, such as the wall of a well, a component of aspot, the stability of a droplet (e.g., a droplet in an emulsion), orthe shell of a capsule. These stimuli are particularly useful ininducing a partition to release its contents. Because a partition may becontained within another partition, and each partition may be responsive(or not responsive) to different stimuli, stimuli-responsiveness may beemployed to release the contents of one partition (e.g., a partitionresponsive to the stimulus) into another partition (e.g., a partitionnot responsive to that stimulus, or less responsive to that stimulus).

In some cases, the contents of an inner capsule may be released into thecontents of an outer capsule by applying a stimulus that dissolves theinner capsule, resulting in a capsule containing a mixed sample. Ofcourse, this embodiment is purely illustrative, andstimuli-responsiveness may be used to release the contents of anysuitable partition into any other suitable partition, medium, orcontainer (see, e.g., Table 1 for more specific examples of partitionswithin partitions).

Examples of stimuli that may be used include chemical stimuli, bulkchanges, biological stimuli, light, thermal stimuli, magnetic stimuli,addition of a medium to a well, and any combination thereof, asdescribed more fully below. (See, e.g., Esser-Kahn et al., (2011)Macromolecules 44: 5539-5553; Wang et al., (2009) ChemPhysChem10:2405-2409.)

a. Chemical Stimuli and Bulk Changes

Numerous chemical triggers may be used to trigger the disruption ofpartitions (e.g., Plunkett et al., Biomacromolecules, 2005, 6:632-637).Examples of these chemical changes may include, but are not limited topH-mediated changes to the integrity of a component of a partition,disintegration of a component of a partition via chemical cleavage ofcrosslink bonds, and triggered depolymerization of a component of apartition. Bulk changes may also be used to trigger disruption ofpartitions.

A change in pH of a solution, such as a decrease in pH, may triggerdisruption of a partition via a number of different mechanisms. Theaddition of acid may cause degradation or disassembly a portion of apartition through a variety of mechanisms. Addition of protons maydisassemble cross-linking of polymers in a component of a partition,disrupt ionic or hydrogen bonds in a component of a partition, or createnanopores in a component of a partition to allow the inner contents toleak through to the exterior. A change in pH may also destabilize anemulsion, leading to release of the contents of the droplets.

In some examples, a partition is produced from materials that compriseacid-degradable chemical cross-linkers, such a ketals. A decrease in pH,particular to a pH lower than 5, may induce the ketal to convert to aketone and two alcohols and facilitate disruption of the partition. Inother examples, the partitions may be produced from materials comprisingone or more polyelectrolytes that are pH sensitive. A decrease in pH maydisrupt the ionic- or hydrogen-bonding interactions of such partitions,or create nanopores therein. In some cases, partitions made frommaterials comprising polyelectrolytes comprise a charged, gel-based corethat expands and contracts upon a change of pH.

Disruption of cross-linked materials comprising a partition can beaccomplished through a number of mechanisms. In some examples, apartition can be contacted with various chemicals that induce oxidation,reduction or other chemical changes. In some cases, a reducing agent,such as beta-mercaptoethanol, can be used, such that disulfide bonds ofa partition are disrupted. In addition, enzymes may be added to cleavepeptide bonds in materials forming a partition, thereby resulting in aloss of integrity of the partition.

Depolymerization can also be used to disrupt partitions. A chemicaltrigger may be added to facilitate the removal of a protecting headgroup. For example, the trigger may cause removal of a head group of acarbonate ester or carbamate within a polymer, which in turn causesdepolymerization and release of species from the inside of a partition.

In yet another example, a chemical trigger may comprise an osmotictrigger, whereby a change in ion or solute concentration in a solutioninduces swelling of a material used to make a partition. Swelling maycause a buildup of internal pressure such that a partition ruptures torelease its contents. Swelling may also cause an increase in the poresize of the material, allowing species contained within the partition todiffuse out, and vice versa.

A partition may also be made to release its contents via bulk orphysical changes, such as pressure induced rupture, melting, or changesin porosity.

b. Biological Stimuli

Biological stimuli may also be used to trigger disruption of partitions.Generally, biological triggers resemble chemical triggers, but manyexamples use biomolecules, or molecules commonly found in living systemssuch as enzymes, peptides, saccharides, fatty acids, nucleic acids andthe like. For example, partitions may be made from materials comprisingpolymers with peptide cross-links that are sensitive to cleavage byspecific proteases. More specifically, one example may comprise apartition made from materials comprising GFLGK (SEQ ID NO: 34) peptidecross links. Upon addition of a biological trigger such as the proteaseCathepsin B, the peptide cross links of the shell well are cleaved andthe contents of the capsule are released. In other cases, the proteasesmay be heat-activated. In another example, partitions comprise acomponent comprising cellulose. Addition of the hydrolytic enzymechitosan serves as biologic trigger for cleavage of cellulosic bonds,depolymerization of component of the partition comprising chitosan, andrelease of its inner contents.

c. Thermal Stimuli

Partitions may also be induced to release their contents upon theapplication of a thermal stimulus. A change in temperature can cause avariety changes to a partition. A change in heat may cause melting of apartition such that a portion of the partition disintegrates, ordisruption of an emulsion. In other cases, heat may increase theinternal pressure of the inner components of a partition such that thepartition ruptures or explodes. In still other cases, heat may transforma partition into a shrunken dehydrated state. Heat may also act uponheat-sensitive polymers used as materials to construct partitions.

In one example, a partition is made from materials comprising athermo-sensitive hydrogel. Upon the application of heat, such as atemperature above 35 C, the hydrogel material shrinks. The suddenshrinkage of the material increases the pressure and ruptures thepartition.

In some cases, a material used to produce a partition may comprise adiblock polymer, or a mixture of two polymers, with different heatsensitivities. One polymer may be particularly likely to shrink afterthe application of heat, while the other is more heat-stable. When heatis applied to such shell wall, the heat-sensitive polymer may shrink,while the other remains intact, causing a pore to form. In still othercases, a material used to produce a partition may comprise magneticnanoparticles. Exposure to a magnetic field may cause the generation ofheat, leading to rupture of the partition.

d. Magnetic Stimuli

Inclusion of magnetic nanoparticles in a material used to produce apartition may allow triggered rupture of the partition, as describedabove, as well as enable guidance of these partitions to otherpartitions (e.g., guidance of capsules to wells in an array). In oneexample, incorporation of Fe₃O₄ nanoparticles into materials used toproduce partitions triggers rupture in the presence of an oscillatingmagnetic field stimulus.

e. Electrical and Light Stimuli

A partition may also be disrupted as the result of electricalstimulation. Similar to the magnetic particles described in the previoussection, electrically sensitive particles can allow for both triggeredrupture of partitions, as well as other functions such as alignment inan electric field or redox reactions. In one example, partitions madefrom materials comprising electrically sensitive material are aligned inan electric field such that release of inner reagents can be controlled.In other examples, electric fields may induce redox reactions within apartition that may increase porosity.

A light stimulus may also be used to disrupt the partitions. Numerouslight triggers are possible and may include systems that use variousmolecules such as nanoparticles and chromophores capable of absorbingphotons of specific ranges of wavelengths. For example, metal oxidecoatings can be used to produce certain partitions. UV irradiation ofpartitions coated with SiO2/TiO2 may result in disintegration of thepartition wall. In yet another example, photo switchable materials suchas azobenzene groups may be incorporated in the materials used toproduce the partitions. Upon the application of UV or visible light,chemicals such as these undergo a reversible cis-to-trans isomerizationupon absorption of photons. In this aspect, incorporation of photoswitches results in disintegration of a portion of a partition, or anincrease in porosity of a portion of a partition.

f. Application of Stimuli

The devices, methods, compositions, systems, and kits of this disclosuremay be used in combination with any apparatus or device that providessuch trigger or stimulus. For example, if the stimulus is thermal, adevice may be used in combination with a heated or thermally controlledplate, which allows heating of the wells and may induce the rupture ofcapsules. Any of a number of methods of heat transfer may be used forthermal stimuli, including but not limited to applying heat by radiativeheat transfer, convective heat transfer, or conductive heat transfer. Inother cases, if the stimulus is a biological enzyme, the enzyme may beinjected into a device such that it is deposited into each well. Inanother aspect, if the stimulus is a magnetic or electric field, adevice may be used in combination with a magnetic or electric plate.

IX. Applications

a. Polynucleotide Sequencing

Generally, the methods and compositions provided herein are useful forpreparation of polynucleotide fragments for downstream applications suchas sequencing. Sequencing may be performed by any available technique.For example, sequencing may be performed by the classic Sangersequencing method. Sequencing methods may also include: high-throughputsequencing, pyrosequencing, sequencing-by-synthesis, single-moleculesequencing, nanopore sequencing, sequencing-by-ligation,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), next generation sequencing, single molecule sequencing bysynthesis (SMSS) (Helicos), massively-parallel sequencing, clonal singlemolecule Array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing,primer walking, and any other sequencing methods known in the art.

In some cases varying numbers of fragments are sequenced. For example,in some cases about 30%-90% of the fragments are sequenced. In somecases, about 35%-85%, 40%-80%, 45%-75%, 50%-70%, 55%-65%, or 50%-60% ofthe fragments are sequenced. In some cases, at least about 30%, 40%,50%, 60%, 70%, 80%, or 90% of the fragments are sequenced. In some casesless than about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fragmentsare sequenced.

In some cases sequences from fragments are assembled to provide sequenceinformation for a contiguous region of the original targetpolynucleotide that is longer than the individual sequence reads.Individual sequence reads may be about 10-50, 50-100, 100-200, 200-300,300-400, or more nucleotides in length.

The identities of the barcode tags may serve to order the sequence readsfrom individual fragments as well as to differentiate betweenhaplotypes. For example, during the partitioning of individualfragments, parental polynucleotide fragments may separated intodifferent partitions. With an increase in the number of partitions, thelikelihood of a fragment from both a maternal and paternal haplotypecontained in the same partition becomes negligibly small. Thus, sequencereads from fragments in the same partition may be assembled and ordered.

b. Polynucleotide Phasing

This disclosure also provides methods and compositions to preparepolynucleotide fragments in such a manner that may enable phasing orlinkage information to be generated. Such information may allow for thedetection of linked genetic variations in sequences, including geneticvariations (e.g., SNPs, mutations, indels, copy number variations,transversions, translocations, inversions, etc.) that are separated bylong stretches of polynucleotides. The term “indel” refers to a mutationresulting in a colocalized insertion and deletion and a net gain or lossin nucleotides. A “microindel” is an indel that results in a net gain orloss of 1 to 50 nucleotides. These variations may exist in either a cisor trans relationship. In a cis relationship, two or more geneticvariations exist in the same polynucleotide or strand. In a transrelationship, two or more genetic variations exist on multiplepolynucleotide molecules or strands.

Methods provided herein may be used to determine polynucleotide phasing.For example, a polynucleotide sample (e.g., a polynucleotide that spansa given locus or loci) may be partitioned such that at most one moleculeof polynucleotide is present per partition. The polynucleotide may thenbe fragmented, barcoded, and sequenced. The sequences may be examinedfor genetic variation. The detection of genetic variations in the samesequence tagged with two different bar codes may indicate that the twogenetic variations are derived from two separate strands of DNA,reflecting a trans relationship. Conversely, the detection of twodifferent genetic variations tagged with the same bar codes may indicatethat the two genetic variations are from the same strand of DNA,reflecting a cis relationship.

Phase information may be important for the characterization of apolynucleotide fragment, particularly if the polynucleotide fragment isderived from a subject at risk of, having, or suspected of a having aparticular disease or disorder (e.g., hereditary recessive disease suchas cystic fibrosis, cancer, etc.). The information may be able todistinguish between the following possibilities: (1) two geneticvariations within the same gene on the same strand of DNA and (2) twogenetic variations within the same gene but located on separate strandsof DNA. Possibility (1) may indicate that one copy of the gene is normaland the individual is free of the disease, while possibility (2) mayindicate that the individual has or will develop the disease,particularly if the two genetic variations are damaging to the functionof the gene when present within the same gene copy. Similarly, thephasing information may also be able to distinguish between thefollowing possibilities: (1) two genetic variations, each within adifferent gene on the same strand of DNA and (2) two genetic variations,each within a different gene but located on separate strands of DNA.

c. Sequencing Polynucleotides from Small Numbers of Cells

Methods provided herein may also be used to prepare polynucleotidescontained within cells in a manner that enables cell-specificinformation to be obtained. The methods enable detection of geneticvariations (e.g., SNPs, mutations, indels, copy number variations,transversions, translocations, inversions, etc.) from very smallsamples, such as from samples comprising about 10-100 cells. In somecases, about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cells maybe used in the methods described herein. In some cases, at least about1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cells may be used in themethods described herein. In other cases, at most about 5, 10, 20, 30,40, 50, 60, 70, 80, 90 or 100 cells may be used in the methods describedherein.

In an example, a method comprises partitioning a cellular sample (orcrude cell extract) such that at most one cell (or extract of one cell)is present per partition, lysing the cells, fragmenting thepolynucleotides contained within the cells by any of the methodsdescribed herein, attaching the fragmented polynucleotides to barcodes,pooling, and sequencing.

As described elsewhere herein, the barcodes and other reagents may becontained within a partition (e.g., a capsule). These capsules may beloaded into another partition (e.g., a well) before, after, orconcurrently with the loading of the cell, such that each cell iscontacted with a different capsule. This technique may be used to attacha unique barcode to polynucleotides obtained from each cell. Theresulting tagged polynucleotides may then be pooled and sequenced, andthe barcodes may be used to trace the origin of the polynucleotides. Forexample, polynucleotides with identical barcodes may be determined tooriginate from the same cell, while polynucleotides with differentbarcodes may be determined to originate from different cells.

The methods described herein may be used to detect the distribution ofoncogenic mutations across a population of cancerous tumor cells. Forexample, some tumor cells may have a mutation, or amplification, of anoncogene (e.g., HER2, BRAF, EGFR, KRAS) in both alleles (homozygous),others may have a mutation in one allele (heterozygous), and stillothers may have no mutation (wild-type). The methods described hereinmay be used to detect these differences, and also to quantify therelative numbers of homozygous, heterozygous, and wild-type cells. Suchinformation may be used, for example, to stage a particular cancerand/or to monitor the progression of the cancer and its treatment overtime.

In some examples, this disclosure provides methods of identifyingmutations in two different oncogenes (e.g., KRAS and EGFR). If the samecell comprises genes with both mutations, this may indicate a moreaggressive form of cancer. In contrast, if the mutations are located intwo different cells, this may indicate that the cancer is more benign,or less advanced.

d. Analysis of Gene Expression

Methods of the disclosure may be applicable to processing samples forthe detection of changes in gene expression. A sample may comprise acell, mRNA, or cDNA reverse transcribed from mRNA. The sample may be apooled sample, comprising extracts from several different cells ortissues, or a sample comprising extracts from a single cell or tissue.

Cells may be placed directly into a partition (e.g., a microwell) andlysed. After lysis, the methods of the invention may be used to fragmentand barcode the polynucleotides of the cell for sequencing.Polynucleotides may also be extracted from cells prior to introducingthem into a partition used in a method of the invention. Reversetranscription of mRNA may be performed in a partition described herein,or outside of such a partition. Sequencing cDNA may provide anindication of the abundance of a particular transcript in a particularcell over time, or after exposure to a particular condition.

The methods presented above provide several advantages over currentpolynucleotide processing methods. First, inter-operator variability isgreatly reduced. Second, the methods may be carried out in microfluidicdevices, which have a low cost and can be easily fabricated. Third, thecontrolled fragmentation of the target polynucleotides allows the userto produce polynucleotide fragments with a defined and appropriatelength. This aids in partitioning the polynucleotides and also reducesthe amount of sequence information loss due to the present ofoverly-large fragments. The methods and systems also provide a facileworkflow that maintains the integrity of the processed polynucleotide.Additionally, the use of restriction enzymes enables the user to createDNA overhangs (“sticky ends”) that may be designed for compatibilitywith adapters and/or barcodes.

e. Partitioning of Polynucleotides, Such as Chromosomes, from Cells

In one example the methods, compositions, systems, devices, and kitsprovided in this disclosure may be used to partition polynucleotides,including whole chromosomes, from cells. In one example, a single cellor a plurality of cells (e.g., 2, 10, 50, 100, 1000, 10000, 25000,50000, 100000, 500000, 1000000, or more cells) is loaded into a vesselwith lysis buffer and proteinase K, and incubated for a specified periodof time. Utilization of a plurality of cells will enable polynucleotidephasing, for example, by partitioning each polynucleotide to be analyzedin its own partition.

After incubation, the cell lysate is partitioned, for example by flowfocusing the cell lysate into a capsule. If phasing is to be performed,flow focusing is performed such that each capsule comprises only asingle analyte (e.g., a single chromosome), or only a single copy of anyparticular chromosome (e.g., one copy of a first chromosome and one copyof a second chromosome). In some cases, a plurality of chromosomes maybe encapsulated within the same capsule, so long as the chromosomes arenot the same chromosome. The encapsulation is performed under gentleflow, to minimize shearing of the polynucleotides. The capsule may beporous, to allow washing of the contents of the capsule, andintroduction of reagents into the capsule, while maintaining thepolynucleotides (e.g., chromosomes) within the capsules. Theencapsulated polynucleotides (e.g., chromosomes) may then be processedaccording to any of the methods provided in this disclosure, or known inthe art. The capsule shells protect the encapsulated polynucleotides(e.g., chromosomes) from shearing and further degradation. Of course,this method can also be applied to any other cellular component.

As described above, the capsule shell may be used to protect apolynucleotide from shearing. However, a capsule may also be used as apartition to enable compartmentalized shearing of a polynucleotide orother analyte. For example, in some cases a polynucleotide may beencapsulated within a capsule and then subject to ultrasonic shear, orany other suitable shearing. The capsule shell may be configured toremain intact under the shear, while the encapsulated polynucleotide maybe sheared, but will remain within the capsule. In some cases, ahydrogel droplet may be used to accomplish the same end.

f. Cancer Mutation Detection and Forensics

Barcoding methods via amplification-based barcoding schemes inpartitions described herein may be useful generating barcode librariesfrom degraded samples such as, for example, fixed formalin-fixed,paraffin-embedded (FFPE) tissue sections. Methods described herein maybe capable of identifying that all amplicons within a partitionoriginated from the same initial molecule. Indeed, with partitionbarcoding, information can be retained about a unique startingpolynucleotide. Such identification may aid in determinations of librarycomplexity as amplicons from different original molecules can bedistinguished. Moreover, methods described herein can permit assessingunique coverage which may aid in determining variant callingsensitivity. These advantages may be particularly useful in cancermutation detection and forensics.

g. Low Input DNA Applications (Circulating Tumor Cell (CTC) Sequencing)

Barcoding methods described herein may be useful in low polynucleotideinput applications, such as, for example the sequencing of nucleic acidsof circulating tumor cells (CTCs). For example, MALBAC methods describedherein within a partition may aid obtaining good data quality in lowpolynucleotide input applications and/or aid in filtering outamplification errors.

VIII. Kits

In some cases, this disclosure provides kits comprising reagents for thegeneration of partitions. The kit may comprise any suitable reagents andinstructions for the generation of partitions and partitions withinpartitions.

In one example, a kit comprises reagents for generating capsules withindroplets in an emulsion. For example, a kit may comprise reagents forgenerating capsules, reagents for generating an emulsion, andinstructions for introducing the capsules into the droplets of theemulsion. As specified throughout this disclosure, any suitable speciesmay be incorporated into the droplets and/or into the capsule. A kit ofthis disclosure may also provide any of these species, such as apolynucleotide comprising a barcode that is pre-partitioned. Similarly,as described throughout the disclosure, the capsule may be designed torelease its contents into the droplets of the emulsion upon theapplication of a stimulus.

In another example, a kit comprises reagents for generating capsuleswithin capsules. For example, a kit may comprise reagents for generatinginner capsules, reagents for generating outer capsules, and instructionsfor generating capsules within capsules. As specified throughout thisdisclosure, any suitable species may be incorporated into the innerand/or outer capsules. A kit of this disclosure may also provide any ofthese species, such as a polynucleotide comprising a barcode that ispre-partitioned. Similarly, as described throughout the disclosure, theinner capsule may be designed to release its contents into the outercapsule upon the application of a stimulus.

IX. Devices

In some cases, this disclosure provides devices comprising partitionsfor the processing of analytes. A device may be a microwell array, or amicro spot array, as described elsewhere in this disclosure. A devicemay formed in a manner that it comprises any suitable partition. In somecases, a device comprises a plurality of wells, or a plurality of spots.Of course, any partition in a device may also hold other partitions,such as a capsule, a droplet in an emulsion, and the like.

A device may be formed from any suitable material. In some examples, adevice is formed from a material selected from the group consisting offused silica, soda lime glass, borosilicate glass, poly(methylmethacrylate), sapphire, silicon, germanium, cyclic olefin copolymer,polyethylene, polypropylene, polyacrylate, polycarbonate, plastic, andcombinations thereof.

In some cases, a device comprises channels for the flow of fluids intoand between partitions. Any suitable channels may be used. A device maycomprise a fluid inlet and a fluid outlet. The inlet and outlet may beattached to liquid handling devices to introduce species into thedevice. The device may be sealed, before or after introduction of anyspecies.

Materials that are hydrophilic and/or hydrophobic may be used indifferent parts of the device. For example, in some cases a device ofthis disclosure comprises a partition with an interior surfacecomprising a hydrophilic material. In some cases a surface exterior tothe partitions comprises a hydrophobic material. In some cases, a fluidflow path is coated with a hydrophobic or hydrophilic material.

As will be appreciated, the instant disclosure provides for the use ofany of the compositions, libraries, methods, devices, and kits describedherein for a particular use or purpose, including the variousapplications, uses, and purposes described herein. For example, thedisclosure provides for the use of the compositions, methods, libraries,devices, and kits described herein in partitioning species, inpartitioning oligonucleotides, in stimulus-selective release of speciesfrom partitions, in performing reactions (e.g., ligation andamplification reactions) in partitions, in performing nucleic acidsynthesis reactions, in barcoding nucleic acid, in preparingpolynucleotides for sequencing, in sequencing polynucleotides, inpolynucleotide phasing, in sequencing polynucleotides from small numbersof cells, in analyzing gene expression, in partitioning polynucleotidesfrom cells, in mutation detection, in neurologic disorder diagnostics,in diabetes diagnostics, in fetal aneuploidy diagnostics, in cancermutation detection and forensics, in disease detection, in medicaldiagnostics, in low input nucleic acid applications, such as circulatingtumor cell (CTC) sequencing, in a combination thereof, and in any otherapplication, method, process or use described herein.

EXAMPLES Example 1: Production of a Library of Forked AdaptersComprising Barcode Sequences by Asymmetric PCR and Addition of aPartially Complementary Universal Sequence

This example provides methods for the manufacture of forked adapterscomprising barcode sequences compatible with next generation sequencingtechnologies (e.g., ILLUMINA). In this example, the barcode is placed inposition 207 as depicted in FIG. 2.

With reference to FIG. 4, a single-stranded adapter-barcodepolynucleotide sequence 401 comprising a first immobilization region402, a barcode region 403, and a first sequencing primer region 404 issynthesized. The barcode region 403 is a seven nucleotide randomsequence synthesized by including equimolar concentrations of A, G, T,and C in each coupling step.

Following synthesis, the single-stranded adapter-barcode polynucleotide401 is diluted into aqueous droplets in a water-in-oil emulsion suchthat each droplet comprises, on average, 0.1 polynucleotides. Thedroplets also comprise reagents for amplification of the single-strandedadapter-barcode polynucleotide 401, by asymmetric PCR (e.g., polymerase,primers, dNTPs, buffer, salts) and a DNA intercalating dye (e.g.,ethidium bromide). The reverse primer is present in excess of theforward primer, or vice versa, enabling asymmetric amplification. Thepolynucleotides are amplified and the reaction proceeds through anexponential phase of amplification 410, which produces double-strandedproducts 405, and a linear phase amplification 411, which producessingle-stranded products 406.

The droplets are sorted on a fluorescence assisted cell sorter (FACS)412 to collect droplets comprising amplified polynucleotides. Apartially complementary universal sequence 407 is added to thepartitions to generate a partially annealed fork structure 413.Partially complementary universal sequence 407 comprises a secondimmobilization region 408 and a second sequencing primer region 409, thelatter of which comprises a T overhang that is compatible with the Aoverhang on a polynucleotide target to be sequenced (not shown).

Example 2: Fragmentation and Barcoding with Fragmentase

A single-stranded adapter-barcode polynucleotide sequence (e.g., FIG. 4:401) comprising a first immobilization region 402, a barcode region 403,and a first sequencing primer region 404 is synthesized, partitioned,amplified, and sorted as described in Example 1, or by any other methoddescribed in this disclosure. Interfacial polymerization is performed onthe droplet comprising the single-stranded adapter-barcodepolynucleotide sequence, to generate a plurality of capsules comprisinga library of single-stranded adapter-barcode polynucleotide sequences406, where each (or most) sequences in the library differ in thesequence of their respective barcode regions 403. Thus, a library ofencapsulated single-stranded adapter-barcode polynucleotides isgenerated.

Two mixtures are prepared. Mixture Z1 comprises a target polynucleotide(i.e., a polynucleotide to be fragmented and barcoded), a fragmentaseenzyme (e.g., NEBNEXT DSDNA FRAGMENTASE), and a partially complementaryuniversal sequence (e.g., FIG. 4: 407). A second mixture Z2 comprisesthe library of encapsulated single-stranded adapter-barcodepolynucleotides, generated as described above and magnesium chloride ina concentration sufficient to activate the fragmentase enzyme. MixtureZ1, Z2, or both Z1 and Z2 also comprise T4 polymerase, Taq polymerase,and a thermostable ligase.

Mixtures Z1 and Z2 are combined and a capsule within a capsule is formedaccording to methods described elsewhere in this disclosure, such asflow focusing. FIG. 5 illustrates a capsule within a capsule producedaccording to the method described above. The outer capsule 501 comprisesan inner capsule 502 and medium 504. The inner capsule 502 is one memberof the library of encapsulated single-stranded adapter-barcodepolynucleotides. Thus, inner capsule 502 comprises multiple copies of asingle-stranded adapter-barcode polynucleotide 503, which can be used toattach the same barcode to a polynucleotide within a partition, such asouter capsule 501.

The medium 504 contains the contents of mixtures Z1 and Z2, describedabove. More specifically, medium 504 comprises target polynucleotide505, the partially complementary universal sequence 506, and the enzymemix 507 comprising fragmentase, T4 polymerase, Taq polymerase,thermostable ligase, magnesium chloride, and appropriate buffers.

Upon generation of the capsule within capsule, and exposure of thecapsule within capsule to appropriate conditions, the enzymes processthe target polynucleotide. More specifically, the fragmentase fragmentsthe target polynucleotide and the T4 polymerase blunts the ends of thefragmented target polynucleotide. The fragmentase and T4 polymerase arethen heat inactivated and a stimulus is used to rupture inner capsule502, releasing its contents into outer capsule 501. The Taq polymeraseadds 3′-A overhangs to the fragmented, blunt-ended targetpolynucleotide. The single-stranded adapter-barcode polynucleotide 503hybridizes with the partially complementary universal sequence 506,forming a forked adapter with a 3′-T overhang that is compatible withthe 3′-A overhang on the fragmented target polynucleotide. Thethermostable ligase ligates the forked adapter to the fragmented targetpolynucleotide, generating barcoded target polynucleotide. The outercapsule 501 is then ruptured, samples from all outer capsules arepooled, and the target polynucleotides are sequenced. Additionalpreparation steps (e.g., bulk amplification, size selection, etc.) maythen be performed as needed prior to sequencing.

In some cases, mixture Z1 comprises multiple versions of the partiallycomplementary universal sequence 506, where each version has its ownsample-specific barcode.

Moreover, although the example described above utilizes a thermallystable ligase to attach the forked adapter comprising the barcodesequence to the target polynucleotide, PCR can also be used toaccomplish this step, as described elsewhere in this disclosure.

Example 3: Fragmentation and Barcoding by Sonication

A library of encapsulated single-stranded adapter-barcodepolynucleotides is generated as described in Example 2, or by any othersuitable method described in this disclosure. Target polynucleotides(i.e., polynucleotides to be fragmented) are partitioned into capsules.The capsules comprising the target polynucleotides are configured towithstand ultrasonic stress. The capsules comprising the targetpolynucleotides are exposed to ultrasonic stress (e.g., COVARISFocused-Ultrasonicator) and the target polynucleotide is fragmented,generating fragmented target polynucleotide capsules.

A mixture Z1 is prepared, comprising the library of encapsulatedsingle-stranded adapter-barcode polynucleotides (e.g., FIG. 4:406), thefragmented target polynucleotide capsules, a partially complementaryuniversal sequence (e.g., FIG. 4:407), an enzyme mixture (T4 polymerase,Taq polymerase, and a thermostable ligase), and appropriate buffers. Acapsule within capsule is generated according to the method describedelsewhere in this disclosure, such as flow focusing.

FIG. 6 illustrates capsules within a capsule produced according to themethods described above. The outer capsule 601 comprises a plurality ofinner capsules 602 and 605 and medium 604. The inner capsules 602 and605 include capsules comprising single-stranded adapter-barcodepolynucleotides 603 and capsules comprising fragmented targetpolynucleotide 606, respectively. Inner capsule 602 comprises multiplecopies of a single-stranded adapter-barcode polynucleotide 603, whichcan be used to attach the same barcode to a polynucleotide within apartition, such as the fragmented polynucleotides 606 contained withininner capsules 605.

The medium 604 contains the contents of mixture Z1, described above.More specifically, medium 604 comprises a partially complementaryuniversal sequence 607, an enzyme mixture (T4 polymerase, Taqpolymerase, and a thermostable ligase) 608, and appropriate buffers.

Inner capsules 605 comprising fragmented target polynucleotides 606 areexposed to a stimulus to rupture them and release their contents intothe contents of outer capsule 601. The T4 polymerase blunts the ends ofthe fragmented target polynucleotides; the Taq polymerase adds 3′-Aoverhangs to the fragmented, blunt-ended target polynucleotide. The T4polymerase and Taq polymerase is then heat-inactivated and a stimulus isapplied to release the contents of inner capsule 602 into outer capsule601. The single-stranded adapter-barcode polynucleotide 603 hybridizeswith the partially complementary universal sequence 607, forming aforked adapter with a 3′-T overhang that is compatible with the 3′-Aoverhang on the fragmented target polynucleotide. The thermostableligase ligates the forked adapter to the fragmented targetpolynucleotide, generating a barcoded target polynucleotide. The outercapsule 601 is then ruptured, samples from all outer capsules arepooled, and the target polynucleotides are sequenced.

As described in Example 2, in some cases Z1 can comprise multipleversions of the partially complementary universal sequence 607.Furthermore, although this example demonstrates barcoding of a targetpolynucleotide by utilizing a thermostable ligase, PCR can also be usedto accomplish this step.

Example 4: Generation of Forked Adapters by Single Primer IsothermalAmplification (SPIA) and Restriction Digestion

This example demonstrates the synthesis of a forked adapter by SPIA andrestriction digestion. FIG. 7 provides an example of a product (orintermediate) that may be generated according to the methods of thisexample. With reference to FIG. 7, a hairpin adapter 701 (SEQ ID NO: 2)is shown that can be used as a precursor to a forked-adapter asdescribed elsewhere in this disclosure. In this example, the hairpinadapter is synthesized as a single-stranded amplification productutilizing SPIA. The hairpin adapter 701 comprises a double-strandedregion 702, a 3′-T overhang for AT ligation 703, and a region that canbe cut by a restriction enzyme 704 (i.e., between positions 33 and 34).The hairpin adapter may comprise a barcode region and functionalregions, such as immobilization regions and regions for annealing ofsequencing primers.

Cutting of the adapter (e.g., between positions 33 and 34) generates theforked-adapter depicted in FIG. 8a (SEQ ID NOs: 3-4). The adapter is cutby introducing an oligonucleotide sequence complementary to the regionto be cut and exposing the annealed adapter to a restriction enzyme.Ligation of the forked-adapter region depicted in FIG. 8a to a targetpolynucleotide results in the structure depicted in FIG. 8b (SEQ ID NOs:5-6). With reference to FIG. 8, the underlined portions of the sequencesin FIG. 8b comprise the target polynucleotide with 3′-A overhangscompatible for ligation with the forked adapter depicted in FIG. 8a .The sequences shown in FIG. 8b (SEQ ID NOs: 5-6) are then amplified bypolymerase chain reaction to produce SEQ ID NO: 7 (amplification productof SEQ ID NO: 5) and SEQ ID NO: 8 (amplification product of SEQ ID NO:6), shown in FIG. 8c . In FIG. 8c , SEQ ID NO: 7 represents anamplification product of SEQ ID NO: 5 that adds a first immobilizationsequence (underlined 5′ portion) and a second immobilization sequence(underlined 3′ portion) to SEQ ID NO: 5. SEQ ID NO: 8 represents anamplification product of SEQ ID NO: 6 that replaces the unhybridizedportions of SEQ ID NO: 6 with different sequences (underlined 3′ portionand underlined 5′ portion). Additionally, SEQ ID NO: 8 includes a sixnucleotide barcode (TAGTGC; bolded) within the 5′ unhybridized region ofthe polynucleotide. The amplification product therefore comprisesbarcoded target polynucleotide sequence (represented by 111),immobilization sequences, and a barcode.

Example 5: Additional Forked Adapters by Single Primer IsothermalAmplification (SPIA) and Restriction Digestion

This example demonstrates the synthesis of a forked adapter as depictedin FIG. 9a (SEQ ID NOs: 9-10) by SPIA and restriction digestion, where Nrepresents A, T, G, or C. FIG. 9b shows the forked-adapter insingle-stranded format (SEQ ID NO: 11), where the single stranded formatis capable of forming a hairpin structure. Cutting the hairpin structureat the position designated by the asterisk yields the forked adaptershown in FIG. 9 a.

The template for the SPIA will be the sequence shown in FIG. 9c (SEQ IDNO: 12). In FIG. 9c , “R” represents a region of RNA. FIG. 9d shows thehairpin structure formed by the sequence in FIG. 9c . The sequence inFIG. 9d (SEQ ID NO: 12) is treated with polymerase to add nucleotides tothe 3′ end, generating the sequence shown in FIG. 9e (SEQ ID NO: 13).The sequence in FIG. 9e (SEQ ID NO: 13) is then treated with RNase H,which degrades RNA hybridized to DNA, yielding the sequence in FIG. 9f(SEQ ID NO: 14).

Strand displacement SPIA is then performed on SEQ ID NO: 14. The primerin the strand displacement amplification is of the form RRRRRRRRRRRRR(i.e., R₁₃). This primer is an RNA primer that is one base longer thanthe unhybridized 3′ terminus of SEQ ID NO: 14 (i.e., N₁₂) (FIG. 9f ).More specifically, as shown in FIG. 9f , the 3′ terminus of SEQ ID NO:14 contains twelve N nucleotides. The RNA primer contains 13nucleotides. Nucleotides 2-13 of the RNA primer are complementary withthe twelve unhybridized N nucleotides of SEQ ID NO: 14. Nucleotide 1 ofthe RNA primer is complementary with the first hybridized base (goingfrom 3′ to 5′), in this case, T. The RNA primer displaces the A andgenerates the double-stranded extension product shown in FIG. 9g (SEQ IDNOs: 15-16). Because only one primer is present, the reaction producesmultiple copies of the single-stranded product. The single-strandedamplification products are treated with RNase H to generate thesingle-stranded amplification products shown in FIG. 9h (SEQ ID NO: 17).FIG. 9i shows this sequence in 5′-3′ format (SEQ ID NO: 17). FIG. 9jshows this sequence in hairpin format (SEQ ID NO 17).

The hairpin adapter shown in FIG. 9j is then ligated to a fragmentedpolynucleotide with a 3′-A overhang. The hairpin is cleaved between theA and C residues separated by the curved line in FIG. 9j by adding anoligonucleotide complementary to that region and cutting with arestriction enzyme. This generates a forked adapter. PCR amplificationis then conducted, as described in Example 4, to add immobilizationregions and barcodes to the forked adapter that is attached to thetarget polynucleotide.

Example 6: Generation of Forked Adapters Comprising Barcodes byExponential PCR and Hybridization

This example demonstrates the production of forked adapters comprisingbarcodes by hybridization. FIG. 10a shows the exemplary forked adapterprovided in FIG. 8a . As described in Example 4, this adapter may beligated to a target polynucleotide and then an amplification reactionmay be performed to add additional functional sequences, including abarcode. However, a barcode (and other functional sequences) may also beincorporated directly into the forked adapter, prior to attachment ofthe forked adapter to the target polynucleotide. For example, FIG. 10bshows the forked adapter of FIG. 10a , with the addition of a firstimmobilization region (underlined) and a seven nucleotide barcode region(bold/underline; “N”).

The barcoded forked adapter of FIG. 10b is produced by firstsynthesizing SEQ ID NO: 18 as a single strand. The diversity in thebarcode region is generated using an equimolar mixture of A, G, T, andC, as described throughout this disclosure. Droplet-based PCR isperformed, as described in Example 1. However, one DNA primer and oneRNA primer are used to amplify SEQ ID NO: 18 in the droplets. Theamplification is conducted in the presence of an intercalating dye, anddroplets comprising amplified SEQ ID NO: 18 are isolated, as describedin Example 1. FIG. 10c shows the double-stranded amplification product.The underlined portion of SEQ ID NO: 19 is an RNA strand derived fromthe RNA primer. The sequences shown in FIG. 10c are then treated withRNase H, which digests the underlined RNA region, yielding the constructshown in FIG. 10d . In order to generate a forked construct, a partiallycomplementary universal sequence (SEQ ID NO: 21) is added to theconstruct shown in FIG. 10d , producing the product shown in FIG. 10e .The advantage to utilizing this process is that it utilizes thesignificantly greater amplification of polynucleotides provided byexponential PCR versus the linear amplification of polynucleotidesprovided by SPIA.

Example 7: Dual Indexing Approach

This example demonstrates an approach for synthesis of barcodes fordual-index reads. A dual-index read is a read of both strands of adouble-stranded fragment, using barcodes attached to each strand. FIG.11 shows an example of the synthesis of barcodes for a dual-indexingapproach and an example use of the barcodes in a capsules in a capsuleconfiguration.

As shown in FIG. 11a , a first single-stranded adapter-barcodepolynucleotide sequence 1101 comprising a first immobilization region1102, a first barcode region 1103, and a first sequencing primer region1104 is synthesized. In parallel, as shown in FIG. 11b , a secondsingle-stranded adapter-barcode polynucleotide sequence 1131, comprisinga second immobilization region 1132, a second barcode region 1133, and asecond sequencing primer region 1134 is synthesized. In some cases,barcode regions 1103 and 1133 are of the same sequence. In other cases,barcode regions 1103 and 1133 are of different sequences or of partiallydifferent sequences.

Following synthesis, the single-stranded adapter-barcode polynucleotides1101 (FIG. 11a ) and 1131 (FIG. 11b ) are, in parallel, diluted intoaqueous droplets in a water-in-oil emulsion. The droplets also comprisereagents for amplification of the single-stranded adapter-barcodespolynucleotide 1101 (FIGS. 11a ) and 1131 (FIG. 11b ) respectively, byasymmetric PCR (e.g., polymerase, primers, dNTPs, buffer, salts) and aDNA intercalating dye (e.g., ethidium bromide). The reverse primer ispresent in excess of the forward primer, or vice versa, enablingasymmetric amplification. The polynucleotides 1101 (FIGS. 11a ) and 1131(FIG. 11b ) are amplified and the reaction proceeds through anexponential phase of amplification 1110, which produces double-strandedproducts 1105 (FIGS. 11a ) and 1135 (FIG. 11b ), and a linear phaseamplification 1111, which produces single-stranded products 1106 (FIGS.11a ) and 1136 (FIG. 11b ) respectively.

The droplets are sorted on a fluorescence assisted cell sorter (FACS)1112 to collect droplets comprising amplified polynucleotides.

Interfacial polymerization is then performed on the droplets comprisingthe single-stranded adapter-barcode polynucleotide sequences 1106 and1136 droplets respectively, to generate two types of capsules 1120(FIGS. 11a ) and 1150 (FIG. 11b ), each comprising one ofsingle-stranded adapter barcode polynucleotide sequences 1106 or 1136respectively.

Two mixtures are prepared. Mixture Z1 comprises a target polynucleotide(i.e., a polynucleotide to be fragmented and barcoded) 1170 and afragmentase enzyme (e.g., NEBNEXT DSDNA FRAGMENTASE). A second mixtureZ2 comprises capsules 1120 and 1180, generated as described above andmagnesium chloride in a concentration sufficient to activate thefragmentase enzyme. Mixture Z1, Z2, or both Z1 and Z2 also comprise T4polymerase, Taq polymerase, and a thermostable ligase.

Mixtures Z1 and Z2 are combined and a capsule within a capsule is formedaccording to methods described elsewhere in this disclosure, such asflow focusing. FIG. 11c illustrates capsules within a capsule producedaccording to the method described above. The outer capsule 1160comprises capsules 1120 and 1150 and medium 1190. Thus, capsules 1120and 1150 each comprise multiple copies of single-strandedadapter-barcode polynucleotides 1106 and 1136, respectively, and can beused to attach barcodes 1103 and 1133 to a polynucleotide within apartition, such as target polynucleotide 1170 in medium 1190 of outercapsule 1160.

The medium 1190 contains the contents of mixtures Z1 and Z2, describedabove. More specifically, medium 1190 comprises target polynucleotide1170 and the enzyme mix 1180 comprising fragmentase, T4 polymerase, Taqpolymerase, thermostable ligase, magnesium chloride, and appropriatebuffers.

Upon generation of the capsules within a capsule, and exposure of thecapsules within the capsule to appropriate conditions, the enzymesprocess the target polynucleotide. More specifically, the fragmentasefragments the target polynucleotide and the T4 polymerase blunts theends of the fragmented target polynucleotide. The fragmentase and T4polymerase are then heat inactivated and a stimulus is used to rupturecapsules 1120 and 1150, releasing their contents into medium 1190 ofouter capsule 1160. The Taq polymerase adds 3′-A overhangs to thefragmented, blunt-ended target polynucleotide. The single-strandedadapter-barcode polynucleotide 1106 hybridizes with single-strandedadapter-barcode polynucleotide 1136, forming a forked adapter,comprising barcode regions 1103 and 1133, with a 3′-T overhang that iscompatible with the 3′-A overhang (not shown) on the fragmented targetpolynucleotide. The thermostable ligase ligates the forked adapter tothe fragmented target polynucleotide, generating barcoded targetpolynucleotide. The outer capsule 1160 is then ruptured, samples fromall outer capsules are pooled, and the target polynucleotides aresequenced.

Moreover, although the example described above utilizes a thermallystable ligase to attach the forked adapter comprising the barcodesequence to the target polynucleotide, PCR can also be used toaccomplish this step, as described elsewhere in this disclosure.

Example 8: Production of a Forked Adapter Comprising Barcode Sequencesby Bead Emulsion PCR and Addition of a Partially Complementary UniversalSequence

As shown in FIG. 14a , a single-stranded adapter-barcode sequence 1401comprising a first immobilization region 1402, a barcode region 1403,and a first sequencing primer region 1404 is synthesized. Followingsynthesis, the single-stranded adapter-barcode sequence 1401 is dilutedinto aqueous droplets in a water-in-oil emulsion such that each dropletcomprises, on average, 1 polynucleotide. The droplets also comprisefirst beads 1405 that are linked, via a photolabile linker, to one ormore copies of an RNA primer 1406 complementary to a sequence comprisedin the first sequencing primer region 1404; a DNA primer complementaryto a sequence (not shown) comprised in the first immobilization region1402; and reagents necessary for amplification (e.g., polymerase, dNTPs,buffer, salts) of single-stranded adapter-barcode sequence 1401. Thepolynucleotides are amplified 1407 which produces double-strandedproducts 1408 both attached to the first beads 1405 to form structure1420 and in solution (not shown).

The emulsion is then broken and the emulsion components are pooled toform a product mixture. As shown in FIG. 14b , the liberated beads arethen washed 1409 (via centrifugation) several times with appropriatemedium, treated with sodium hydroxide (NaOH) 1410 to denature thedouble-stranded products attached to the first beads 1405, and thenfurther washed 1411. After denaturation 1410 and washing 1411 ofstructure 1420, the resulting structure 1430 comprises a single-strandedcomplement 1412 to the single-stranded adapter-barcode sequence 1401,comprising a complementary immobilization region 1413, a complementarybarcode region 1414, and a complementary sequencing primer region 1415.As shown, the complementary sequencing primer region 1415 comprises theRNA primer 1406. Structure 1430 is then resuspended in an appropriatemedium.

Next, as shown in FIG. 14c , second beads 1416 that comprise one or morecopies of a DNA polynucleotide 1417 complementary to the complementaryimmobilization region 1413 are then added to the medium. Via thecomplementary DNA polynucleotide 1417 and the complementaryimmobilization region 1413 of the single-stranded complement 1412, thesecond beads 1416 bind to the single-stranded complement 1412. Thesingle stranded complement is now bound at one end to first bead 1405and at its other end second bead 1416 to form structure 1440.

As shown in FIG. 14d , structure 1440 is then centrifuged 1418 using aglycerol gradient to separate structure 1440 from structure 1430 notcomprised in structure 1440. In cases where the second beads 1416 aremagnetic, a magnetic separation may be used as an alternative. Theproduct is then treated with NaOH 1419 to denature the single-strandedcomplement 1412 from the second bead 1416, resulting in regeneration ofstructure 1430. Structure 1430 is then subject to several rounds ofwashing (via centrifugation) to remove second beads 1416.Single-stranded complement 1412, attached to structure 1430, representsa single-stranded barcode adapter.

As shown in FIG. 14e , single-stranded complement 1412 can be used togenerate a forked adapter. To generate a forked adapter 1450, thesingle-stranded complement 1412 is then released 1424 from structure1430 with light and then combined 1425 with a universal complementarysequence 1426 or is first combined 1425 with a universal complementarysequence 1426 and then released 1424 from structure 1430. In order togenerate ligatable ends, RNAase H is used to digest the RNA primer 1406of the single-stranded complement 1412 and a Type IIs restriction enzymeis used to generate a single base T overhang on the universalcomplementary sequence 1426. The T overhang is compatible with the Aoverhang on a polynucleotide target to be sequenced (not shown).

Example 9: Production of a Forked Adapter Comprising Barcode Sequencesby Bead Emulsion PCR and Addition of a Partially Complementary UniversalSequence

As shown in FIG. 15a , a single-stranded adapter-barcode sequence 1501comprising a first immobilization region 1502, a barcode region 1503,and a first sequencing primer region 1504 is synthesized. Followingsynthesis, the single-stranded adapter-barcode sequence 1501 is dilutedinto aqueous droplets in a water-in-oil emulsion such that each dropletcomprises, on average, 1 polynucleotides. The droplets also comprisefirst beads 1505 that are linked, via a photolabile linker, to one ormore copies of an RNA primer 1506 complementary to a sequence comprisedin the first immobilization region 1502; a DNA primer complementary to asequence (not shown) comprised in the first sequencing primer region1502; and reagents necessary for amplification (e.g., polymerase, dNTPs,buffer, salts) of single-stranded adapter-barcode sequence 1501. Thepolynucleotides are amplified 1507 which produces double-strandedproducts 1508 both attached to the first beads 1505 to form structure1520 and in solution (not shown).

The emulsion is then broken and the emulsion components are pooled toform a product mixture. As shown in FIG. 15b , the liberated beads arethen washed 1509 (via centrifugation) several times with appropriatemedium, treated with sodium hydroxide (NaOH) 1510 to denature thedouble-stranded products attached to the first beads 1505, and thenfurther washed 1511. After denaturation 1510 and washing 1511 ofstructure 1520, the resulting structure 1430 comprises a single-strandedcomplement 1512 to the single-stranded adapter-barcode sequence 1501,comprising a complementary immobilization region 1513, a complementarybarcode region 1514, and a complementary sequencing primer region 1515.As shown, the complementary sequencing primer region 1515 comprises theRNA primer 1506. Structure 1530 is then resuspended in an appropriatemedium.

Next, as shown in FIG. 15c , second beads 1516 that comprise one or morecopies of a DNA polynucleotide 1517 complementary to the complementarysequencing primer region 1515 are then added to the medium. Via thecomplementary DNA polynucleotide 1517 and the complementary sequencingprimer region 1515 of the single-stranded complement 1512, the secondbeads 1416 bind to the single-stranded complement 1512. The singlestranded complement is now bound at one end to first bead 1505 and atits other end second bead 1516 to form structure 1540.

As shown in FIG. 15d , structure 1540 is then centrifuged 1518 using aglycerol gradient to separate structure 1540 from structure 1530 notcomprised in structure 1540. In cases where the second beads 1516 aremagnetic, a magnetic separation may be used as an alternative. Theproduct is then treated with NaOH 1519 to denature the single-strandedcomplement 1512 from the second bead 1516, resulting in regeneration ofstructure 1530. Structure 1530 is then subject to several rounds ofwashing (via centrifugation) to remove second beads 1516.Single-stranded complement 1512, attached to structure 1530, representsa single-stranded barcode adapter.

As shown in FIG. 15e , single-stranded complement 1512 can be used togenerate a forked adapter. To generate a forked adapter 1550, thesingle-stranded complement 1512 is then optionally released 1524 fromstructure 1530 with light and then combined 1525 with a universalcomplementary sequence 1526. In order to generate ligatable ends, a TypeIIs restriction enzyme is used to generate a single base T overhang onthe universal complementary sequence 1526. The T overhang is compatiblewith the A overhang on a polynucleotide target to be sequenced (notshown).

Example 10: Production of a Forked Adapter Template Barcode Sequences byBead Emulsion PCR and an Adapter Derived Therefrom

As shown in FIG. 16, structure 1600 comprising a magnetic bead(1601)-bound single-stranded adapter-barcode sequence 1602 is producedaccording to methods described in Example 8, Example 9, or any othermethod described herein. Next, structure 1600 is partitioned intocapsules (or alternatively, another emulsion) 1620 by methods describedherein, for example, interfacial polymerization. The capsules 1620 alsocomprise reagents for amplification of the single-strandedadapter-barcode sequence 1602, by asymmetric PCR (e.g., polymerase,primers, dNTPs, buffer, salts). The reverse primer is present in excessof the forward primer, or vice versa, enabling asymmetric amplification.Single-stranded adapter-barcode sequence 1602 is amplified 1603 and thereaction proceeds through a linear phase amplification 1604, whichproduces single-stranded adapter product 1605, complementary tosingle-stranded barcode adapter-template 1602. At this juncture,capsules 1620 comprise both single-stranded adapter 1605 in solution andmagnetic bead (1601)-bound single-stranded adapter-barcode sequence1602. Capsules 1620 are then separated from those not comprising beads(and thus templates 1602 and single-stranded adapters 1605) by magneticseparation 1606. Capsules 1620 may be ruptured and forked adaptersgenerated as described in Example 9.

Example 11: Barcoding with Bead Emulsion PCR and Fragmentation withFragmentase

As shown in FIG. 17, structure 1700 comprising a magnetic bead(1701)-bound single-stranded adapter-barcode sequence 1702 is producedaccording to methods described in Example 8, Example 9, or any othermethod described herein. Interfacial polymerization is performed on thedroplet comprising the structure 1700, to generate a capsule 1704comprising single-stranded adapter-barcode sequence 1702 attached, via aphotolabile linker, to a bead 1701.

Two mixtures are prepared. Mixture Z1 comprises a target polynucleotide(i.e., a polynucleotide to be fragmented and barcoded), a fragmentaseenzyme (e.g., NEBNEXT DSDNA FRAGMENTASE), and a partially complementaryuniversal sequence. A second mixture Z2 comprises capsule 1704 generatedas described above and magnesium chloride in a concentration sufficientto activate the fragmentase enzyme. Mixture Z1, Z2, or both Z1 and Z2also comprise T4 polymerase, Taq polymerase, and a thermostable ligase.

Mixtures Z1 and Z2 are combined and a capsule within a capsule is formedaccording to methods described elsewhere in this disclosure, such asflow focusing. FIG. 17 illustrates a capsule within a capsule producedaccording to the method described above. The outer capsule 1703comprises an inner capsule 1704 and medium 1705. The inner capsule 1704is one member of a library of encapsulated, bead-bound single-strandedbarcode adapters. Thus, inner capsule 1704 comprises multiple copies ofstructure 1700, which can be used to generate a free single-strandedadapter-barcode sequence 1702 and attach the same barcode adapter to apolynucleotide within a partition, such as outer capsule 1703.

The medium 1705 contains the contents of mixtures Z1 and Z2, describedabove. More specifically, medium 1705 comprises target polynucleotide1706, the partially complementary universal sequence 1707, and theenzyme mix 1708 comprising fragmentase, T4 polymerase, Taq polymerase,thermostable ligase, magnesium chloride, and appropriate buffers.

Upon generation of the capsule within capsule, and exposure of thecapsule within capsule to appropriate conditions, the enzymes processthe target polynucleotide. More specifically, the fragmentase fragmentsthe target polynucleotide and the T4 polymerase blunts the ends of thefragmented target polynucleotide. The fragmentase and T4 polymerase arethen heat inactivated and a stimulus is used to rupture inner capsule1704, releasing its contents into outer capsule 1703. The Taq polymeraseadds 3′-A overhangs to the fragmented, blunt-ended targetpolynucleotide. The single-stranded adapter-barcode sequence 1702hybridizes with the partially complementary universal sequence 1707 andis released from the bead with light, forming a forked adapter with a3′-T overhang that is compatible with the 3′-A overhang on thefragmented target polynucleotide. The thermostable ligase ligates theforked adapter to the fragmented target polynucleotide, generatingbarcoded target polynucleotide. The outer capsule 1703 is then ruptured,samples from all outer capsules are pooled, and the targetpolynucleotides are sequenced. Additional preparation steps (e.g., bulkamplification, size selection, etc.) may be performed as needed prior tosequencing.

In some cases Z1 can comprise multiple versions of the partiallycomplementary universal sequence 1707. Furthermore, although thisexample demonstrates barcoding of a target polynucleotide by utilizing athermostable ligase, PCR can also be used to accomplish this step.

Example 12: Barcoding with Bead Emulsion PCR and Fragmentation bySonication

As shown in FIG. 18, structure 1800 comprising a magnetic bead(1801)-bound single-stranded adapter-barcode sequence 1802 is producedaccording to methods described in Example 8, Example 9, or any othermethod described herein. Interfacial polymerization is performed on thedroplet comprising the structure 1800, to generate a capsule 1803comprising single-stranded adapter-barcode sequence 1802 attached, via aphotolabile linker, to a bead 1801. Target polynucleotides (i.e.,polynucleotides to be fragmented) are partitioned into capsules 1804.The capsules 1804 comprising the target polynucleotides are configuredto withstand ultrasonic stress. The capsules 1804 comprising the targetpolynucleotides are exposed to ultrasonic stress (e.g., COVARISFocused-Ultrasonicator) and the target polynucleotide is fragmented,generating fragmented target polynucleotide capsules.

A mixture Z1 is prepared, comprising capsule 1803, the fragmented targetpolynucleotide capsules 1804, a partially complementary universalsequence 1805, an enzyme mixture (T4 polymerase, Taq polymerase, and athermostable ligase) 1806, and appropriate buffers. A capsule withincapsule is generated according to the method described elsewhere in thisdisclosure, such as flow focusing.

FIG. 18 illustrates capsules within a capsule produced according to themethods described above. The outer capsule 1807 comprises capsules 1803and 1804 and medium 1808. The inner capsules 1803 and 1804 includecapsules comprising structure 1800 and capsules comprising fragmentedtarget polynucleotide 1809, respectively. Inner capsule 1803 comprisesmultiple copies of structure 1800, which can be used to generate a freesingle-stranded barcode adapter 1802 and attach the same barcode adapterto a polynucleotide within a partition, such as the fragmentedpolynucleotides 1809 contained within inner capsules 1804.

The medium 1808 contains the contents of mixture Z1, described above.More specifically, medium 1808 comprises a partially complementaryuniversal sequence 1805, an enzyme mixture (T4 polymerase, Taqpolymerase, and a thermostable ligase) 1806, and appropriate buffers.

Inner capsules 1804 comprising fragmented target polynucleotides 1809are exposed to a stimulus to rupture them and release their contentsinto the contents of outer capsule 1807. The T4 polymerase blunts theends of the fragmented target polynucleotides; the Taq polymerase adds3′-A overhangs to the fragmented, blunt-ended target polynucleotide. TheT4 polymerase and Taq polymerase is then heat-inactivated and a stimulusis applied to release the contents of inner capsule 1803 into outercapsule 1807. The single-stranded adapter-barcode sequence 1802hybridizes with the partially complementary universal sequence 1805 andthe adapter is released from the bead with light forming a forkedadapter with a 3′-T overhang that is compatible with the 3′-A overhangon the fragmented target polynucleotide. The thermostable ligase ligatesthe forked adapter to the fragmented target polynucleotide, generating abarcoded target polynucleotide. The outer capsule 1807 is then ruptured,samples from all outer capsules are pooled, and the targetpolynucleotides are sequenced.

In some cases Z1 can comprise multiple versions of the partiallycomplementary universal sequence 1807. Furthermore, although thisexample demonstrates barcoding of a target polynucleotide by utilizing athermostable ligase, PCR can also be used to accomplish this step.

Example 13: Barcoding with Multiple Annealing and Looping-BasedAmplification (MALBAC)

As shown in FIG. 19a , a primer comprising SEQ ID NO: 36 is prepared.The primer comprises a barcode region (designated “Barcode”), a primersequencing region (designated “PrimingSeq”), and a eight-nucleotidevariable region (designated as “NNNNNNNN”) that may comprise anycombination of A, T, C, or G. The primer shown in FIG. 19 is combinedwith a target polynucleotide (indicated by the loop in FIG. 19), alongwith a polymerase (e.g., Vent, exo+DeepVent, exo−DeepVent) possessing ofstrand-displacement activity into a partition (e.g., a capsule, dropletof an emulsion, etc.). In some cases, a non strand-displacing polymerase(e.g., Taq, PfuUltra) is used. The partition is then subject to MALBACamplification. Appropriate MALBAC cycling conditions are known and are,described for example, in Zong et al., Science, 338(6114), 1622-1626(2012), which is incorporated herein by reference, in its entirety.

A looped MALBAC product is produced as shown in FIG. 19b as SEQ ID NO:23. The looped MALBAC product comprises the original primer shown inFIG. 19a , the target polynucleotide to be barcoded oriented in a loop,and a region complementary to and hybridized to the original primersequence. The partition is broken and the contents recovered. In somecases, a plurality of partitions are generated. The partitions arecollectively broken, the contents of each recovered, and then pooled.

Next, the generated MALBAC product shown in FIG. 19b is treated with arestriction enzyme (e.g., BfuCl or similar) to generate a 4-basepairoverhang (in this case, GATC shown in italics) on the MALBAC product.This structure is represented by SEQ ID NO: 24 and shown in FIG. 19c . Aforked adapter, shown in FIG. 19d as SEQ ID NO: 25 and SEQ ID NO: 37,comprising an overhang (in this case, CTAG shown in bold) complementaryto the overhang generated on the MALBAC product. The forked adapter ismixed with the MALBAC product in FIG. 19c and the complementary regionshybridize. A thermostable ligase is used to ligate the forked adapterand MALBAC product together to form the desired structure FIG. 19e asSEQ ID NO: 26. Additional amplification methods (e.g., PCR) can be usedto add additional regions (e.g., immobilization regions, additionalbarcodes, etc.) to the forked adapter.

In some cases, other basepair overhangs (e.g., 1 basepair overhang-10basepair overhang) may be desired. Restriction enzymes used to generatethese overhangs may be used as an alternative, including those describedherein, where desired. In one example, a two basepair overhang isgenerated on the MALBAC product using Taq^(α)I.

As an alternative, the primer shown in FIG. 19a can be designed suchthat an RNA primer sequence is placed 5′ of the barcode region, suchthat an RNAase is used to generate an overhang. As shown in FIG. 19f ,MALBAC product 1900 comprises an RNA primer sequence 1901 placed 5′ of abarcode region 1902. MALBAC product 1900 also comprises a sequencingprimer region 1903, the target polynucleotide 1904, a complementarysequencing primer region 1905, a complementary barcode region 1906, anda region 1907 complementary to the RNA primer sequence 1901. MALBACproduct 1900 is treated with an RNAse H 1908 and the RNA primer regionsequence 1901 is digested to yield a 2-6 basepair overhang 1909 onMALBAC product 1900 to give structure 1920. A universal complementaryregion 1910 is then added to structure 1910 that comprises a regioncomplementary to the overhang on structure 1910. Universal complementaryregion 1910 then hybridizes with structure 1920 and a thermostableligase is used to ligate universal complementary region 1910 tostructure 1920.

Example 14: Barcoding with Multiple Annealing and Looping-BasedAmplification (MALBAC)

As shown in FIG. 20, a template 2000 comprising a barcode region iscombined with agents 2001 necessary for PCR into a capsule 2002, using,for example interfacial polymerization or any other method describedherein. PCR is used to generate a MALBAC primer from the template 2000.Next, the capsule 2000 is encapsulated into an outer capsule 2003 thatalso comprises a mixture 2004 that comprises a target polynucleotide2005 to be barcoded and reagents 2006 necessary for MALBAC amplification(e.g., DeepVent polymerase, dNTPs, buffer). Capsule 2002 is broken uponproper exposure of capsule 2002 to a stimulus designed to rupturecapsule 2002, the contents of capsule 2002 mix with those of mixture2004. MALBAC amplification of the target polynucleotide 2005 commencesto produce a MALBAC product similar to that described as that shown as1900 in FIG. 19 f.

The outer capsule 2003 is then broken with the appropriate stimulus andthe contents recovered. The MALBAC product is then treated with anappropriate restriction enzyme and coupled to a forked adapter in amatter as described in Example 13. Additional downstream preparationsteps (e.g., bulk amplification, size selection, etc.) are thenperformed as needed.

Example 15: Barcoding with Multiple Annealing and Looping-BasedAmplification (MALBAC)

As shown in FIG. 21a , a MALBAC primer 2100 is prepared. MALBAC primer2100 comprises a sequence priming region 2101 and an 8-nucleotidevariable region 1902. Primer 2100 is combined with target polynucleotide2103, along with a polymerase (e.g., Vent, exo+DeepVent, exo−DeepVent)possessing of strand-displacement activity into a partition (e.g., acapsule, emulsion, etc.). In some cases, a non strand-displacingpolymerase (e.g., Taq, PfuUltra) is used. The partition is then subjectto MALBAC amplification 2104.

A looped MALBAC product 2110 is produced and comprises sequencingpriming region 2101, target polynucleotide 2103, and a complementarysequence priming region 2105. Shown in linear form 2120 in FIG. 21b ,MALBAC product 2110 is then contacted with another primer 2130 thatcomprises a sequencing primer region 2106, a barcode region 2107, and animmobilization region 2108. Primer 2130 is produced using asymmetricdigital PCR. Using a single cycle of PCR, the primer is used to generatedouble-stranded product 2140 that comprises primer 2130, and, thus,barcode region 2107.

Double-stranded product 2140 may be then denatured and subsequentlycontacted with another primer 2150 shown in FIG. 21c . Primer 2150comprises a barcode region 2109, a sequencing primer region 2111, and animmobilization region 2112. In the presence of primers 2113 and 2114,additional rounds of PCR can add the barcode region 2109 to the end ofthe target polynucleotide that attached to barcode region 2107.Additional downstream preparation steps (e.g., bulk amplification, sizeselection, etc.) are then performed as needed.

Example 16: Barcoding with Multiple Annealing and Looping-BasedAmplification (MALBAC)

As shown in FIG. 22, a primer template 2200 comprising a barcode regionis combined with agents 2201 necessary for PCR into a capsule 2202,using, for example interfacial polymerization or any other methoddescribed herein. PCR is then used to generate a primer from template2200. Next, the capsule 2200 is encapsulated into an outer capsule 2003that also comprises a mixture 2204 that comprises a targetpolynucleotide 2205 to be barcoded, reagents 2206 necessary for MALBACamplification (e.g., DeepVent polymerase, dNTPs, buffer), and a MALBACprimer 2207 that does not comprise a barcode (similar to MALBAC primer2100 described in Example 15). MALBAC amplification of the targetpolynucleotide 2205 commences to produce a MALBAC product similar tothat described as that shown as 2110 in FIG. 21a . Capsule 2202 is thenbroken upon proper exposure of capsule 2202 to a stimulus designed torupture capsule 2202, the contents of capsule 2202 mix with those ofmixture 2004. A single cycle of PCR commences using the primer generatedfrom template 2200 to generate a barcoded product, similar to thatdescribed in Example 15.

Outer capsule 2203 is then broken with the appropriate stimulus and thecontents recovered. Additional downstream preparation steps (e.g., bulkamplification, size selection, addition of additional barcodes, etc.)are then performed as needed.

Example 17: Barcoding with Transposase and Tagmentation

As shown in FIG. 23, a single-stranded adapter-barcode polynucleotidesequence 2300 is synthesized, partitioned, amplified, and sorted asdescribed in Example 1, or by any other method described in thisdisclosure. Interfacial polymerization is performed on the dropletcomprising the single-stranded adapter-barcode polynucleotide sequence,to generate a capsule 2301.

Two mixtures are prepared. Mixture Z1 comprises a target polynucleotide2302 (i.e., a polynucleotide to be fragmented and barcoded), atransposome 2303, and a partially complementary universal sequence 2304.A second mixture Z2 comprises capsule 2301, generated as described aboveand reagents 2305 necessary for PCR as described elsewhere herein.

Mixtures Z1 and Z2 are combined and a capsule within a capsule is formedaccording to methods described elsewhere in this disclosure, such asflow focusing. FIG. 23 illustrates a capsule within a capsule producedaccording to the method described above. The outer capsule 2306comprises capsule 2301 and medium 2307. Capsule 2301 is one member of alibrary of encapsulated single-stranded adapter-barcode polynucleotides.Thus, capsule 2301 comprises multiple copies of a single-strandedadapter-barcode polynucleotide 2300, which can be used to attach thesame barcode to a polynucleotide within a partition, such as outercapsule 2306.

The medium 2307 contains the contents of mixtures Z1 and Z2, describedabove. More specifically, medium 2307 comprises target polynucleotide2302, the partially complementary universal sequence 2304, and thereagents 2305 necessary for PCR, including a hot start Taq.

Upon generation of the capsule within capsule, and exposure of thecapsule within capsule to appropriate conditions, the transposomeprocess the target polynucleotide. More specifically, the transposasefragments the target polynucleotide via tagmentations and tags it with acommon priming sequence. The tagged target polynucleotide is then heatedto fill in any gap in the target nucleotide generated by thetransposase. The transposase is then heat inactivated and a stimulus isused to rupture inner capsule 2301, releasing its contents into outercapsule 2306. The hot start Taq is activated by heating the outercapsule 2306 to 95° C. The reaction proceeds with limited cycle PCR toadd single-stranded adapter-barcode polynucleotide sequence 2300 totarget polynucleotide 2302. The outer capsule 2306 is then ruptured andthe target polynucleotides are sequenced.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications may be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method comprising: a. providing a targetpolynucleotide; b. fragmenting said target polynucleotide to generate aplurality of non-overlapping first polynucleotide fragments; c.partitioning said first polynucleotide fragments to generate partitionedfirst polynucleotide fragments, wherein at least one partition of saidpartitioned first polynucleotide fragments comprises a firstpolynucleotide fragment with a unique sequence within said at least onepartition; and d. fragmenting said partitioned first polynucleotidefragments to generate a plurality of non-overlapping secondpolynucleotide fragments.
 2. The method of claim 1, further comprising:a. fragmenting said target polynucleotide to generate a plurality ofnon-overlapping third polynucleotide fragments; b. partitioning saidthird polynucleotide fragments to generate partitioned thirdpolynucleotide fragments, wherein at least one partition of saidpartitioned third polynucleotide fragments comprises a thirdpolynucleotide fragment with a unique sequence within said at least onepartition; and c. fragmenting said partitioned third polynucleotidefragments to generate a plurality of non-overlapping fourthpolynucleotide fragments.
 3. The method of claim 2, wherein said thirdpolynucleotide fragments overlap with said first polynucleotidefragments.
 4. The method of claim 2, wherein said fourth polynucleotidefragments overlap with said second polynucleotide fragments.
 5. Themethod of claim 1, wherein said target polynucleotide is selected fromthe group consisting of DNA, RNA, and cDNA.
 6. The method of claim 2,wherein at least one of said first, second, third, and fourthpolynucleotide fragments are generated by an enzyme.
 7. The method ofclaim 6, wherein said enzyme is a restriction enzyme.
 8. The method ofclaim 7, wherein said restriction enzyme used to generate said firstpolynucleotide fragments is different from said restriction enzyme usedto generate said third polynucleotide fragments.
 9. The method of claim7, wherein said restriction enzyme used to generate said secondpolynucleotide fragments is different from said restriction enzyme usedto generate said fourth polynucleotide fragments.
 10. The method ofclaim 7, wherein said restriction enzyme has a recognition site of atleast about six nucleotides in length.
 11. A method for generating aplurality of nucleic acid molecules comprising barcode sequences,comprising: synthesizing a plurality of nucleic acid moleculescomprising a plurality of barcode sequences; separating said pluralityof nucleic acid molecules into a plurality of partitions comprising aplurality of beads, wherein a partition of said plurality of partitionscomprises a single nucleic acid molecule of said plurality of nucleicacid molecules and a single bead of said plurality of beads; in saidpartition, subjecting said single nucleic acid molecule to nucleic acidamplification to generate at least 1,000 nucleic acid molecules attachedto said single bead, wherein said at least 1,000 nucleic acid moleculescomprise a barcode sequence of said plurality of barcode sequences,which barcode sequence is identical for said at least 1,000 nucleic acidmolecules; and recovering said at least 1,000 nucleic acid moleculesattached to said single bead from said partition.
 12. The method ofclaim 11, wherein said single bead is a gel bead.
 13. The method ofclaim 12, wherein said gel bead is a degradable gel bead.
 14. The methodof claim 11, wherein said at least 1,000 nucleic acid molecules arereleasably attached to said single bead.
 15. The method of claim 11,wherein said plurality of barcode sequences comprise 1,000 differentbarcode sequences.
 16. The method of claim 11, wherein said plurality ofbarcode sequences comprise 10,000 different barcode sequences.
 17. Themethod of claim 11, wherein said plurality of barcode sequences comprise100,000 different barcode sequences.
 18. The method of claim 11, whereinsaid plurality of barcode sequences comprise 1,000,000 different barcodesequences.
 19. The method of claim 11, wherein said at least 1,000nucleic acid molecules attached to said single bead is at least 10,000nucleic acid molecules.
 20. The method of claim 11, wherein said atleast 1,000 nucleic acid molecules attached to said single bead is atleast 100,000 nucleic acid molecules.
 21. The method of claim 11,wherein said at least 1,000 nucleic acid molecules attached to saidsingle bead is at least 1,000,000 nucleic acid molecules.
 22. The methodof claim 11, wherein said at least 1,000 nucleic acid molecules attachedto said single bead is at least 10,000,000 nucleic acid molecules. 23.The method of claim 11, wherein each nucleic acid molecule of saidplurality of nucleic acid molecules further comprises an immobilizationsequence and wherein said plurality of beads are coupled to a nucleicacid molecule comprising a sequence complementary to said immobilizationsequence.
 24. The method of claim 23, wherein each nucleic acid moleculeof said plurality of nucleic acid molecules further comprises an adaptersequence capable of coupling to a target nucleic acid molecule.
 25. Themethod of claim 23, wherein each nucleic acid molecule of said pluralityof nucleic acid molecules further comprises a sequencing primersequence.
 26. The method of claim 11, wherein said plurality ofpartitions is a plurality of wells.
 27. The method of claim 11, whereinsaid plurality of partitions is a plurality of aqueous droplets in anemulsion.
 28. The method of claim 11, wherein said separating comprisesperforming a limiting dilution, thereby generating dilutedpolynucleotides.
 29. The method of claim 28, wherein said separatingfurther comprises partitioning said diluted polynucleotides.
 30. Themethod of claim 11, wherein greater than 5% of said plurality ofpartitions comprise a single nucleic acid molecule of said plurality ofnucleic acid molecules that is unique within that partition.